The contribution of anaerobic ammonium oxidation to nitrogen loss in two temperate eutrophic estuaries Catarina Teixeira a, b, * , Catarina Magalhães b , Samantha B. Joye c , Adriano A. Bordalo a, b a Laboratory of Hydrobiology and Ecology, Institute of Biomedical Sciences Abel Salazar (ICBAS-UP), University of Porto, Rua Jorge Viterbo Ferreira, 228, 4050-313 Porto, Portugal b Interdisciplinary Centre of Marine and Environmental Research (CIIMAR/CIMAR), Rua dos Bragas, 289, 4050-123 Porto, Portugal c Department of Marine Sciences, University of Georgia, Athens, GA 30602-3636, USA article info Article history: Received 18 March 2013 Accepted 19 March 2014 Available online 26 March 2014 Keywords: anammox nitrogen sediments estuary abstract Studies of anaerobic ammonium oxidation (anammox) continue to show the significance of this meta- bolic pathway for the removal of nitrogen (N) in several natural environments, including estuaries. However, the seasonal dynamics of the anammox process and related environmental controls within estuarine systems remains poorly explored. We evaluated the seasonal anammox activity along a salinity gradient in two temperate Atlantic estuaries, the Ave and the Douro (NW Portugal). Anammox potential rates were measured in anaerobic sediment slurries using 15 N-labeled NO  3 and NH þ 4 amendments. Production of 29 N 2 and 30 N 2 in the slurries was quantified using membrane inlet mass spectrometry (MIMS). Environmental characteristics of the sediment and water column were also monitored. Anam- mox potentials in the Ave and Douro estuarine sediments varied between 0.8e8.4, and 0e2.9 nmol cm 3 wet sediment h 1 , respectively, with high seasonal and spatial fluctuations. Inorganic nitrogen avail- ability emerged as the primary environmental control of anammox activity, while water temperature appeared to modulate seasonal variations. The contribution of anammox to overall N 2 production averaged over 20%, suggesting that the role of anammox in removing fixed N from these two systems cannot be neglected. Ó 2014 Elsevier Ltd. All rights reserved. 1. Introduction Anaerobic ammonium oxidation (anammox) uses nitrite as the oxidant for anaerobic ammonium oxidation and produces molec- ular nitrogen as a final product via the following stoichiometry (Strous et al., 1998): 1NH þ 4 þ 1:32NO  2 þ 0:66HCO  3 þ 0:13H þ /1:02N 2 þ 0:26NO  3 þ 0:066CH 2 O 0:5 N 0:15 þ 2:03H 2 O The loss of ammonium under anoxic conditions was first iden- tified in the 1960s in seawater (Richards, 1965), and the existence of anammox microorganisms was predicted based on thermodynam- ical calculations (Broda, 1977). The anammox process was subse- quently observed (and patented) in 1994 in wastewater treatment reactors (Mulder et al., 1995). Later on, Thamdrup and Dalsgaard (2002) provided evidence of anammox activity in natural environ- ments. Since then, the anammox process has been demonstrated across a variety of diverse habitats, including, marine (e.g., Dalsgaard et al., 2003; Kuypers et al., 2003), estuarine (e.g., Trimmer et al., 2003; Risgaard-Petersen et al., 2004), freshwater (Schubert et al., 2006), terrestrial (Humbert et al., 2010), low-temperature polar (Rysgaard et al., 2004), high-temperature hot spring and hydro- thermal vents (Byrne et al., 2009; Jaeschke, 2009), oil reservoirs (Li et al., 2010), and in marine sponges (Hoffmann et al., 2009). In estuarine sediments, the percent of N 2 flux due to anammox ranges between 0 and 33% (Rich et al., 2008; Dale et al., 2009; Dong et al., 2009; Nicholls and Trimmer, 2009; Teixeira et al., 2012). Thus, anammox appears to be ecologically important in some estuarine sediments, although its contribution is variable, and measurements are only available for a few systems. Water column nitrate con- centration, nitrite availability in the sediment, organic carbon content, and temperature were reported previously as the main factors regulating anammox in estuarine sediments (Trimmer et al., 2003; Risgaard-Petersen et al., 2004; Meyer et al., 2005; Rich et al., 2008; Nicholls and Trimmer, 2009; Teixeira et al., 2012). * Corresponding author. Laboratory of Hydrobiology and Ecology, Institute of Biomedical Sciences (ICBAS-UP), Rua Jorge Viterbo Ferreira, 228, 4050-313 Porto, Portugal. E-mail address: catarina@icbas.up.pt (C. Teixeira). Contents lists available at ScienceDirect Estuarine, Coastal and Shelf Science journal homepage: www.elsevier.com/locate/ecss http://dx.doi.org/10.1016/j.ecss.2014.03.023 0272-7714/Ó 2014 Elsevier Ltd. All rights reserved. Estuarine, Coastal and Shelf Science 143 (2014) 41e47