Introduction Anaerobic ammonium oxidation (anammox) is the ox- idation of ammonium with nitrite as the electron accep- tor and dinitrogen gas as the product [10]. Anammox catabolism proceeds via hydrazine (N 2 H 4 ) and hydroxyl- amine (NH 2 OH). The reaction is exergenic (deltaG = –358 kJ/mol) and provides the energy for anammox an- abolism, e.g. the fixation of CO 2 with nitrite as the elec- tron donor (leading to the anaerobic production of ni- trate). The process is mediated by obligately anaerobic Candidatus “Scalindua brodae”, sp. nov., Candidatus “Scalindua wagneri”, sp. nov., Two New Species of Anaerobic Ammonium Oxidizing Bacteria Markus Schmid 1 , Kerry Walsh 2 , Rick Webb 3 , W. Irene C. Rijpstra 4 , Katinka van de Pas-Schoonen 5 , Mark Jan Verbruggen 5 , Thomas Hill 2 , Bruce Moffett 2 , John Fuerst 3 , Stefan Schouten 4 , Jaap S. Sinninghe Damsté 4 , James Harris 6 , Phil Shaw 6 , Mike Jetten 5 , and Marc Strous 5 1 Kluyver Laboratory for Biotechnology, Delft University of Technology, Delft, The Netherlands 2 School of Biosciences, University of East London, London 3 Department of Microbiology, University of Queensland, Brisbane, Queensland, Australia 4 Royal Netherlands Institute for Sea Research (NIOZ), Department of Marine Biogeochemistry and Toxicology, Den Burgh, The Netherlands 5 Department of Microbiology, Faculty of Science, University of Nijmegen, Nijmegen, The Netherlands 6 Institute of Water and Environment, Cranfield University, Bedfordshire Received: May 26, 2003 Abstract Anaerobic ammonium oxidation (anammox) is both a promising process in wastewater treatment and a long overlooked microbial physiology that can contribute significantly to biological nitrogen cycling in the world’s oceans. Anammox is mediated by a monophyletic group of bacteria that branches deeply in the Planctomycetales. Here we describe a new genus and species of anaerobic ammonium oxidizing planctomycetes, discovered in a wastewater treatment plant (wwtp) treating landfill leachate in Pitsea, UK. The biomass from this wwtp showed high anammox activity (5.0 ± 0.5 nmol/mg protein/min) and produced hydrazine from hydroxylamine, one of the unique features of anammox bacteria. Eight new planctomycete 16S rRNA gene sequences were present in the 16S rRNA gene clone library generated from the biomass. Four of these were affiliated to known anammox 16S rRNA gene sequences, but branched much closer to the root of the planctomycete line of descent. Fluorescence in situ hybridization (FISH) with oligonucleotide probes specific for these new sequences showed that two species (belonging to the same genus) together made up > 99% of the planctomycete population which constituted 20% of the total microbial community. The identification of these organisms as typical anammox bacteria was confirmed with electron microscopy and lipid analysis. The new species, provisionally named Candida- tus “Scalindua brodae” and “Scalindua wagneri” considerably extend the biodiversity of the anammox lineage on the 16S rRNA gene level, but otherwise resemble known anammox bacteria. Simultaneously, another new species of the same genus, Candidatus “Scalindua sorokinii”, was detected in the water col- umn of the Black Sea, making this genus the most widespread of all anammox bacteria described so far. Key words: ammonium – nitrite – nitrate – 16S rRNA approach chemolithoautotrophic bacteria, two of which have been named provisionally: Candidatus “Brocadia anammoxi- dans” [25] and Candidatus “Kuenenia stuttgartiensis” [22]. These species (and genera) form a monophyletic cluster inside the Planctomycetales, one of the major divi- sions of the Bacteria. Recently it was shown that this order might be the closest to the root of the bacterial do- main [3]. Planctomycetes have a proteinaceous cell wall without peptidoglycan and a differentiated cytoplasm 0723-2020/03/26/04-529 $ 15.00/0 System. Appl. Microbiol. 26, 529–538 (2003) © Urban & Fischer Verlag http://www.urbanfischer.de/journals/sam