LETTERS PUBLISHED ONLINE: 6 JANUARY 2013 | DOI: 10.1038/NGEO1683 Hotspots of anaerobic ammonium oxidation at land–freshwater interfaces Guibing Zhu 1 * , Shanyun Wang 1,2 , Weidong Wang 1 , Yu Wang 1 , Leiliu Zhou 1 , Bo Jiang 1 , Huub J. M. Op den Camp 3 , Nils Risgaard-Petersen 4 , Lorenz Schwark 5 , Yongzhen Peng 2 , Mariet M. Hefting 6 , Mike S. M. Jetten 3 and Chengqing Yin 1 For decades, the conversion of organic nitrogen to dinitrogen gas by heterotrophic bacteria, termed heterotrophic denitrifi- cation, was assumed to be the main pathway of nitrogen loss in natural ecosystems. Recently, however, autotrophic bacteria have been shown to oxidize ammonium in the absence of oxy- gen, yielding dinitrogen gas. This process, termed anammox, accounts for over 50% of nitrogen loss in marine ecosystems 1–5 . However, the significance of anammox in freshwater ecosys- tems has remained uncertain 6,7 . Here, we report the occurrence of anammox hotspots at land–freshwater interfaces of lake riparian zones in North China, using molecular and isotopic tracing technologies. Laboratory incubations measuring anam- mox activity at substrate concentrations no more than 10% of those observed in situ yielded some of the highest potential activities reported for natural environments to date. Potential rates of anammox peaked in sediments sampled from the inter- face between land and water, as did the abundance of annamox bacteria. Scaling our findings up to the entire lake system, we estimate that interfacial anammox hotspots account for the loss of 103 g Nm -2 yr -1 from this lake region, and around one fifth of the nitrogen lost from the land–water interface. The interface between two adjacent ecosystems often contains hot zones for biogeochemical cycles 8 . Riparian zones, transitional boundary zones between terrestrial and aquatic ecosystems, play important roles in regulating landscape-level interactions in the vertical, transversal and longitudinal geometry of three- dimensional space 9 . Furthermore, riparian zones have long been regarded as biogeochemical hotspots of nitrogen cycling 8 with high rates of ammonium oxidation 10 , oxygen consumption 11 and nitrogen loss 12 . In freshwater ecosystems, especially those with intensified allochthonous input, nitrate supply is generally assumed to be the limiting factor for the anammox process 6,13–15 . Riparian zones have long been regarded as nitrate sinks 9 because of the fluctuating hydrological regime and intensive material exchange between inland and water, in particular the oxidized and reduced nitrogen compounds. On the basis of the studies described above and our previous anammox studies in wetland ecosystems 6,13,16 , we put forward the hypothesis that riparian zones are hotspots of anammox activity. The present study aims to investigate the abundance, activity, biodiversity, qualitative and quantitative importance of anammox in riparian zones. Baiyangdian Lake, the largest freshwater lake in 1 Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, PO Box 2871, 18 Shuangqing Road, Haidian District, Beijing 100085, China, 2 State Key Laboratory of Urban Water Resource and Environment, Harbin Institute of Technology, Harbin 150090, China, 3 Department of Microbiology, Radboud University Nijmegen, 3 6525 AJ Nijmegen, The Netherlands, 4 Center for Geomicrobiology, Department of Bioscience, Aarhus University, 8000 Aarhus C, Denmark, 5 Institute for Geosciences, University of Kiel, D-24098 Kiel, Germany, 6 Ecology and Biodiversity Group, Department of Biology, Utrecht University, 3508 TB Utrecht, The Netherlands. *e-mail: gbzhu@rcees.ac.cn. North China with well-developed riparian zones, was selected as the study site (Supplementary Fig. S1). The hypothesis that riparian zones would act as hotspots of anammox was first tested along a single transect, and then further confirmed by extensive large-scale sampling in the whole lake. To investigate the occurrence of anammox hotspots in riparian zones, the presence of anammox bacteria needed to be confirmed first. The 16S rRNA genes of Planctomycetes and anammox bacteria in ten surface (0–5 cm) and ten subsurface (30–60 cm) sediments/soils of landward phase, land–freshwater interface and waterward phase were specifically amplified. All samples except landward soil (site E) harboured anammox 16S rRNA genes which were most closely affiliated to Brocadia species (similarity 97.6– 98.8%; Fig. 1 and Supplementary Fig. S2 and Table S1). To address the occurrence of anammox in particular hotspots, the abundance of anammox bacteria in the riparian zone was estimated using qPCR assays. The highest amount of hydrazine synthase (hzsB) gene was detected in interface sediments (1 × 10 7 copies g −1 , site B), being 2–3 orders of magnitude higher than those observed in the adjacent waterward sediments (site A) and interface soil (site C; Fig. 1), whereas in the landward phase (sites D and E) the anammox abundance was below the detection limit (<10 4 ). Anammox bacterial abundance was observed over a wide range with high spatial heterogeneity, and much higher in surface than subsurface sediment/soil. The highest potential anammox rates were observed at 11–13 nmol N g −1 h −1 in the superficial interface sediments (site B) with the 15 N-tracer technique, significantly higher than those in waterward sediments (1 nmol N g −1 h −1 , site A), inter- face soils (0.8–1.0 nmol N g −1 h −1 , site C) and landward soils (0.05–0.14 nmol N g −1 h −1 , site D; Tukey’s multiple comparisons under analysis of variance, p < 0.001, n = 20; Fig. 1 and Sup- plementary Fig. S3 and Table S1). A similar trend was observed for subsurface sediments/soils. The analysis of variance analyses suggest that seasonal variations are insignificant. Above all, the spatiotemporal analysis revealed that anammox abundance and activity in riparian zone was of high variability and the land–freshwater interface may be an anammox hotspot. To further verify the hypothesis of anammox hotspots at land–freshwater interfaces on the whole lake scale, a further ten waterward and ten interface sediments (0–5 cm) were sampled in February and August 2011. First, 16S rRNA genes analysis NATURE GEOSCIENCE | VOL 6 | FEBRUARY 2013 | www.nature.com/naturegeoscience 103 © 2013 Macmillan Publishers Limited. All rights reserved.