Axiovision software package

Blood smears were prepared on slides, air-dried and fixed with methanol for 1 min at room temperature. Then, the slides were blocked with PBS (pH 7.2) plus 10% FBS overnight. After three washes with PBS, indirect immunofluorescence staining was performed, using anti-BmSA1 antiserum as the primary antibody and Alexa Fluor 594-conjugated goat anti-mouse IgG (Life Technologies, Inc., Rockville, MD, United States) as the secondary antibody, and nucleus was stained with Hoechst (Beyotime Biotechnology, Shanghai, China). Next, the samples were visualized first under a confocal microscope (Olympus Life Science, Tokyo, Japan) from a normal perspective and then under a confocal laser microscope (Olympus Life Science, Tokyo, Japan) from a three-dimensional perspective. Image analysis was performed using an Axiovision software package (Carl Zeiss Jena, Oberkochen, Germany).

The medium fed to the stable enrichment culture was changed in 2014 by removing nitrate and increasing trace element concentrations (see above). In 2016, after approximately 2 years since the change in medium composition, a biomass sample of 1.5 mL was pelleted for 3 min at 4000 G. The pellet was then washed twice with 1 ml phosphate-buffered saline (PBS: 130 mM NaCl and 10 mM phosphate buffer pH 7.4). After two washing steps, the sample was pelleted, re-suspended in 300 μL of PBS and mixed with 900 μL 4% paraformaldehyde for fixation (1:3 ratio). The fixation sample was kept mixing overnight at 10 RPM at a temperature of 4°C. Probes used were S-^∗-DBACT-1027-a-A-18 for ‘Ca. Methylomirabilis’ spp. and S-^∗-AAA-FW-641 for ‘Ca. Methanoperedens’ spp. (Ettwig et al., 2016 (link)). Hybridization was performed as described previously (Ettwig et al., 2009 (link)) using 40% formamide. Images were captured with a Zeiss Axioplan 2 microscope equipped with a CCD camera, and processed with the Axiovision software package (Zeiss, Germany).

Biomass samples (2 mL) from the coculture were taken on days 85, 136, and 464 and centrifuged for 5 min at maximum speed. The pellet was washed with 1 mL phosphate-buffered saline (10 mM Na₂HPO₄/NaH₂PO₄ pH 7.5 and 130 mM NaCl) and fixed using paraformaldehyde. Subsequently, FISH was performed as described before (Ettwig et al. 2008 ). A mixture of three probes (EUB338, EUB338II, and EUB338III, targeting most bacteria) was used to visualize the general bacterial population (Amann et al. 1990 (link); Daims et al. 1999 (link)). A general probe targeting most archaea (ARCH0915) was used to visualize archaea (Stahl and Amann 1991 ). To visualize anammox bacteria, Ca.Methylomirabilis-like bacteria, and Ca.Methanoperedens-like archaea, AMX820, DBACT1043, and DBACT1043b mix and DARCH0641 probes were used, respectively (Schmid et al. 2001 , Raghoebarsing et al. 2006 , Ettwig et al. 2009 (link), Schubert et al. 2011 (link)). Probes were Cy3, Cy5, or FLUOS labeled. All samples were counterstained with the DNA stain 4′,6-diamidino-2-phenylindole (DAPI). Slides were examined and images obtained by utilization of a Zeiss Axioplan 2 epifluorescence microscope equipped with a digital camera, in combination with the AxioVision software package (Zeiss, Germany).