Please cite this article in press as: J. Böllmann, et al., Autofluorescent characteristics of Candidatus Brocadia fulgida and the consequences for FISH and microscopic detection, Syst. Appl. Microbiol. (2018), https://doi.org/10.1016/j.syapm.2018.09.002 ARTICLE IN PRESS G Model SYAPM-25947; No. of Pages 10 Systematic and Applied Microbiology xxx (2018) xxx–xxx Contents lists available at ScienceDirect Systematic and Applied Microbiology journal homepage: www.elsevier.de/syapm Autofluorescent characteristics of Candidatus Brocadia fulgida and the consequences for FISH and microscopic detection Jörg Böllmann ∗ , Steffen Engelbrecht, Marion Martienssen Department of Biotechnology for Water Treatment, BTU-Cottbus-Senftenberg, Siemens-Halske-Ring 8, 03046 Cottbus, Germany a r t i c l e i n f o Article history: Received 19 June 2018 Received in revised form 10 August 2018 Accepted 13 September 2018 Keywords: 16S rDNA Hydrazine synthase hzsA Hydrazine oxidase hzo FISH Candidatus Kuenenia stuttgartiensis Candidatus Brocadia fulgida a b s t r a c t An enrichment culture of Candidatus Brocadia fulgida was identified by three independent methods: analysis of autofluorescence using different microscope filter blocks and a fluorescence spectrometer, fluorescence in situ hybridization (FISH) with anammox-specific probes and partial sequencing of the 16S rDNA, hydrazine synthase hzsA and hydrazine oxidoreductase hzo. The filter block BV-2A (400–440, 470 LP, Nikon) was suitable for preliminary detection of Ca. B. fulgida. An excitation-emission matrix revealed three pairs of excitation-emission maxima: 288–330 nm, 288–478 nm and 417–478 nm. Sev- eral autofluorescent cell clusters could not be stained with DAPI or by FISH, suggesting empty but intact cells (ghost cells) or inhibited permeability. Successful staining of autofluorescent cells with the FISH probes Ban162 and Bfu613, even at higher formamide concentrations, suggested insufficient specificity of Ban162. Under certain conditions, Ca. B. fulgida lost its autofluorescence, which reduced the reliabil- ity of autofluorescence for identification and detection. Non-fluorescent Ca. Brocadia cells could not be stained with Ban162, but with Bfu613 at higher formamide concentrations, suggesting a dependency between both parameters. The phylogenetic analysis showed only good taxonomical clustering of the 16S rDNA and hzsA. In conclusion, careful consideration of autofluorescent characteristics is recom- mended when analysing and presenting FISH observations of Ca. B. fulgida to avoid misinterpretations and misidentifications. © 2018 Elsevier GmbH. All rights reserved. Introduction Anammox (anaerobic ammonium oxidizing) bacteria have been reported from many ecosystems and manmade habitats [3,18]. Their anaerobic chemolithoautotrophic equimolar conversion of ammonia and nitrite into nitrogen gas plays an important role in the global nitrogen cycle besides denitrification [23] and might amelio- rate the negative effects of excess anthropogenic nitrogen [15,24]. In several ecosystems, this has been suggested as the main nitrogen sink [37]. The anammox process is a promising tool for removing nitrogen, especially from nitrogen-rich and carbon-poor wastewa- ters [49], although handling is difficult and the growth rate is very slow [23]. Nevertheless, besides many small-scale approaches, the process has been implemented successfully several times in large- scale wastewater treatment plants [49]. The five known Candidatus genera Kuenenia, Scalindua, Broca- dia, Jettenia and Anammoxoglobus are monophyletic and deeply branched from within the Planctomycetes [7,37,45]. Culture- ∗ Corresponding author. E-mail address: boellman@b-tu.de (J. Böllmann). independent methods, such as fluorescence in situ hybridization (FISH), the amplification and sequencing of the 16S rDNA or other anammox-specific genomic regions, such as hydrazine oxidase (hzo) or hydrazine synthase (hzs) targeted by many anammox- specific primer sets [33,32,17], are the most common tools for detection and identification of these bacteria [10]. Hydrazine syn- thase produces hydrazine, a unique anammox intermediate from nitric oxide and ammonium, and it has been used as a specific biomarker for anammox bacteria [16,17,25]. Hydrazine oxidore- ductase is an essential enzyme that dehydrogenates hydrazine to dinitrogen gas [25], and it was the first functional gene used for detecting anammox bacteria [16]. It has been found in many differ- ent types of environments [16,32] and, although many sequences are available, only a few identified sequences have been published [7,9,17,20,26,33,52]. Therefore, genomic data banks still require reliably identified sequences in order to provide sufficient infor- mation for the identification of the rapidly increasing number of shot gun sequences and for detailed phylogenetic analysis. More- over, identification by sequencing is often limited by few database entries of identified strains that have poor coverage [15,52]. For FISH, there are many available probes for different taxo- nomic specificities [45], making this method a powerful tool for https://doi.org/10.1016/j.syapm.2018.09.002 0723-2020/© 2018 Elsevier GmbH. All rights reserved.