FEMS Microbiology Ecology Advance Access published April 17, 2016 MS for FEMS Microbiology Ecology Article Type: Research Article Phylogenetic Diversity and Ecophysiology of Candidate Phylum Saccharibacteria in Activated Sludge Tomonori Kindaichi1, 2, Shiro Yamaoka1, Ryohei Uehara1, Noriatsu Ozaki1, Akiyoshi Ohashi1, Mads Albertsen2, Per Halkjær Nielsen2, and Jeppe Lund Nielsen2, * Downloaded from 1 Department of Civil and Environmental Engineering, Hiroshima University, 1-4-1 Kagamiyama, http://femsec.oxfordjournals.org/ Higashihiroshima, 739-8527 Japan. 2 Center for Microbial Communities, Department of Chemistry and Bioscience, Aalborg University, Fredrik Bajers Vej 7H, DK-9220 Aalborg E, Denmark. by guest on April 22, 2016 Keywords: Saccharibacteria; candidate division TM7; microautoradiography; exo-enzyme activity; activated sludge; FISH Running Title: Ecophysiology of Saccharibacteria in Activated Sludge * Corresponding author: Jeppe Lund Nielsen Center for Microbial Communities, Department of Chemistry and Bioscience, Aalborg University, Fredrik Bajers Vej 7H, DK-9220 Aalborg E, Denmark. E-mail address: [email protected] 1 ABSTRACT Candidate phylum Saccharibacteria (former TM7) are abundant and widespread in nature, but little is known about their ecophysiology and detailed phylogeny. In this study phylogeny, morphology, and ecophysiology of Saccharibacteria were investigated in activated sludge from nine wastewater treatment plants (WWTPs) from Japan and Denmark using the full-cycle 16S rRNA approach and microautoradiography (MAR) - fluorescence in situ hybridization (FISH). Phylogenetic analysis showed that Saccharibacteria from all WWTPs were evenly distributed within subdivision 1 and 3 and in a distinct phylogenetic clade. Three probes were designed for distinct saccharibacterial groups, and Downloaded from revealed morphotypes representing thin filaments, thick filaments, and rods/cocci. MAR-FISH showed that most probe-defined Saccharibacteria utilized glucose under aerobic-, nitrate reducing-, and http://femsec.oxfordjournals.org/ anaerobic conditions. Some Saccharibacteria also utilized N-acetylglucosamine, oleic acid, amino acids, and butyrate, which are not predicted from available genomes so far. In addition, some filamentous Saccharibacteria exhibited β-galactosidase and lipase activities determined using a combination of enzyme-labeled fluorescence and FISH (ELF-FISH). No uptake of acetate, propionate, pyruvate, by guest on April 22, 2016 glycerol, and ethanol was observed. These results indicate that Saccharibacteria is a phylogenetically diverse group and play a role in the degradation of various organic compounds as well as sugar compounds under aerobic-, nitrate reducing-, and anaerobic conditions. One sentence summary Candidatus Saccharibacteria in activated sludge are phylogenetically diverse and utilize oleic acid, amino acids, and N-acetylglucosamine as well as glucose as the carbon sources. 2 INTRODUCTION The microbial life on earth can be divided into at least 67 different phyla (SILVA database, Quast et al. 2013), and approximately half of these remain without any cultured representatives and are thus categorized as “candidate phyla (or divisions)”. Recently, most of the candidate phyla were renamed and some of them proposed as superphyla based on metabolic features predicted by single-cell genomics of two hundred uncultured bacterial and archaeal cells from various habitats (Rinke et al., 2013) or by retrieval of complete or near complete genomes using metagenomics (Albertsen et al., 2013). Although a large number of environmental sequences related to candidate phyla have been obtained from various Downloaded from environments, their ecophysiological roles in complex microbial communities are still largely unknown. The candidate phylum Saccharibacteria (former candidate division TM7, hereafter called http://femsec.oxfordjournals.org/ Saccharibacteria) is a well-described candidate phylum and has been frequently detected in various natural environments (Hugenholtz et al., 2001; Ferrari et al., 2014), human oral cavities (Ouverney et al., 2003; Marcy et al., 2007; Dinis et al., 2011; Soro et al., 2014; He et al., 2015) and activated sludge (Hugenholtz et al. 2001; Thomsen et al., 2002; Albertsen et al., 2013). Based on 16S rRNA gene by guest on April 22, 2016 sequences, three subdivisions within the phylum have been proposed (Hugenholtz et al., 2001), and PCR primers and fluorescence in situ hybridization (FISH) probes targeting these are available. Hugenholtz and coworkers (2001) reported that members within subdivision 1 showed filamentous morphotypes, while members within subdivision 2 and 3 showed non-filamentous (i.e., rods or cocci) morphotypes. The morphotypes of Saccharibacteria with retrieved genomes are described as either rod shaped (Marcy et al., 2007) or small cocci (Albertsen et al., 2013; He et al., 2015) and belong to the subdivision 3. In addition, when an isolated saccharibacterial species was cultivated in dual-species biofilms, they showed a pleomorphic characteristic, which formed long filaments and short rods/cocci depending on the coexisting bacterial species (Soro et al., 2014). Little is known about the detailed phylogeny and physiology of Saccharibacteria in activated sludge, although Saccharibacteria including filamentous morphotype have been frequently detected in 3 activated sludge by FISH analysis (Hugenholtz et al., 2001; Thomsen et al., 2002; Nielsen et al., 2010a; Mielczarek et al., 2012; Nielsen et al., 2012a). Filamentous bacteria are the main cause of bulking problems in the wastewater treatment process as excessive growth results in poor settling of the biomass in the clarifier. However, they also play a pivotal role in floc formation and floc stability (Nielsen et al., 2009). More than 30 different filamentous morphotypes have been identified in wastewater treatment plants (WWTPs) treating municipal wastewater based on conventional light microscopic characterization, chemical staining methods (Eikelboom, 2000) and molecular methods (e.g., FISH and subcloning of 16S rRNA genes). Our current knowledge of the physiology of Saccharibacteria is Downloaded from primarily based on the in situ method microautoradiography (MAR) combined with FISH (MAR-FISH), where the specific metabolic activity of unculturable microorganisms within complex http://femsec.oxfordjournals.org/ microbial communities can be studied at single-cell level (Nielsen and Nielsen, 2005). The previous MAR-FISH studies revealed that filamentous Saccharibacteria can take up several monosaccharides and amino acids (Ariesyady et al., 2007; Nielsen et al., 2009). In addition, filamentous Saccharibacteria have been shown to express protease activity as determined by exoenzyme detection (Nielsen et al., by guest on April 22, 2016 2010b) and seem to be involved in hydrolysis of proteins in activated sludge (Nielsen et al., 2010a). Furthermore, a single-cell genomic study of Saccharibacteria in the human oral cavity indicated that they might be able to use oligosaccharides and amino acids (Marcy et al., 2007). More recently, complete genomes of Saccharibacteria, obtained through metagenomics, suggested that some members have an obligate fermentative metabolism, fermenting glucose and other sugars, while producing lactate (Albertsen et al., 2013; Kantor et al., 2013). However, most in situ studies have been conducted with broad phylogenetic probes (i.e., the phylum specific probe TM7905) and thereby lack the resolution to resolve if the observed traits are general for the phylum or related to specific taxa within Saccharibacteria. Therefore, development of new oligonucleotide probes with higher resolution and application of these in combination with methods that can provide information on their ecophysiology and improve process performance and control of activated sludge bulking problems. 4 In the present study, the full-cycle 16S rRNA approach was combined with ELF-FISH and comprehensive MAR-FISH analysis to identify and characterize members of Saccharibacteria in activated sludge from one Japanese and eight Danish WWTPs. Three FISH probes were designed to enhance the resolution within subdivision 1 and target a distinct clade within the phylum Saccharibacteria, and applied to investigate their abundance, morphology, in situ substrate uptake, and exo-enzyme activity. We performed in situ methods with the phylum level TM7905 probe to screen MAR- or ELF-positive Saccharibacteria. If Saccharibacteria, detected with TM7905 probe, showed MAR-positive signals, then we applied the newly designed probes for detailed analyses. Downloaded from MATERIALS AND METHODS http://femsec.oxfordjournals.org/ Activated sludge samples Activated sludge samples were collected from one Japanese (Higashihiroshima) and eight Danish full-scale WWTPs (Aalborg West, Sønderborg, Randers, Viborg, Ejby Mølle, Horsens, Aars, and Marselisborg), which all had stable operation for several years (Nielsen et al., 2010a). The information by guest on April 22, 2016 of the WWTPs is listed in Table 1. Fresh sludge samples were collected from the aeration tanks in the period from April 2011 and January 2012 and stored at 4°C for up to 24 h before proceeding with the experimental analysis. Samples from three plants (Higashihiroshima, Ejby Mølle, and Marselisborg) were used for the MAR-FISH analyses. Furthermore, the sludge samples from Higashihiroshima and Aalborg West WWTPs were used for studying exoenzyme activity by ELF-FISH. DNA extraction and