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 PCR amplification

DNA was extracted from activated sludge using the Fast DNA spin kit for soil (MP Biomedicals, Irvine,

CA, USA). Extracted DNA was used for the amplification of 16S rRNA gene fragments with a

TM7-specific primer set, TM7580f and univ1492r (Hugenholtz et al., 2001; Lane, 1991). The PCR condition was as follows: 10 min of initial denaturation at 94°C, followed by 30 cycles of 1 min at 94°C,

5 1 min at 60°C, and 2 min at 72°C. The final extension was performed for 5 min at 72°C. PCR products were confirmed using a 1% (w/v) agarose gel.

Cloning, sequencing and phylogenetic analysis

PCR products were purified using a QIAquick PCR Purification Kit (Qiagen, Hilden, Germany). The purified PCR products were ligated into a pCR-XL-TOPO vector and transformed into One Shot E. coli cells according to the manufacturer’s instructions (TOPO XL PCR Cloning Kit; Carlsbad, CA, USA).

Clones were randomly selected and clone libraries from each WWTP were constructed. The 16S rRNA Downloaded from genes were sequenced by Takara Bio (Otsu, Japan) or Macrogen (Amsterdam, The Netherlands). All sequences were checked for chimeric artifacts by the DECIPHER program (Wright et al., 2012). http://femsec.oxfordjournals.org/ Sequences with 97% or higher similarity were grouped into operational taxonomic units (OTUs) using the Distance matrix methods with the similarity correction in the ARB software (Ludwig et al., 2004).

Phylogenetic trees were constructed using the neighbor-joining with jukes-cantor correction model, the maximum parsimony (Phylip DNAPARS), and the maximum likelihood (RAxML) methods using by guest on April 22, 2016 default settings in the ARB software with the MiDAS v1.20 database (McIlroy et al., 2015). A bootstrap resampling analysis for 1000 replicates for three algorithms (neighbor-joining, maximum-parsimony and maximum-likelihood) was conducted using ARB software to estimate the confidence of tree topologies. The 16S rRNA gene sequence data of OTUs obtained in this study were deposited in the

GenBank/EMBL/DDBJ databases under accession numbers AB861987 to AB862089. Putative excreted enzymes were identified in Candidatus Saccharimonas aalborgensis (CP005957) using

PSORTb 3.0 (Yu et al., 2010) with default settings.

FISH and new probe design

Sludge fixation and FISH were performed as described previously (Nielsen, 2009). The oligonucleotide probes used in this study are listed in Table 2. The probes were labeled with Cy3, FLUOS (or Alexa

6 Fluor 488), or Alexa Fluor 647 at the 5' end. Simultaneous hybridizations with the probes requiring different stringency conditions were performed by using a successive hybridization procedure; hybridization with the probe requiring higher stringency was performed first, and then hybridization with the probe requiring lower stringency was performed (Wagner et al., 1994). An Axioimager M1 epifluorescence microscope (Carl Zeiss, Oberkochen, Germany) was used for the identification and quantification by FISH and the exoenzyme activity by ELF-FISH. MAR-FISH observation was performed with a LSM5 PASCAL confocal laser-scanning microscope equipped with an Ar ion laser

(488 nm) and a HeNe laser (543nm) (Carl Zeiss, Oberkochen, Germany). Downloaded from New FISH probes were designed using the probe design tool of the ARB software (Ludwig et al., 2004). The Sacch720a and Sacch720b probes were designed to target members of http://femsec.oxfordjournals.org/ Saccharibacteria belonging to the subdivision 1 with higher detection frequencies in clone libraries as an alternative to TM7305 probe and TM7905 probe for the subdivision 1. Sacch933 probe and the competitor probes were designed to target members of Saccharibacteria which do not hybridize with the

TM7905 probe. The specificity of the probes was confirmed using the program probeCheck (Loy et al., by guest on April 22, 2016 2008) and TestProbe (Quast et al., 2013). In the absence of pure cultures the formamide concentration for optimum stringency of the designed probes were determined by analyzing fluorescence intensities on activated sludge biomass from Higashihiroshima, Aalborg West, Ejby Mølle, or Marselisborg

WWTPs, by applying hybridization buffer containing 0–60% formamide (5% increments). The fluorescence intensities of at least 20 images for each probe were evaluated by image analysis using the

ImageJ software (Collins, 2007). Enrichment cultures of anammox consortia without Saccharibacteria confirmed by pyrosequencing (data not shown) (Kindaichi et al., 2007; Kindaichi et al., 2011) were used as the negative control to check the specificity of the new probes. The specificity of the designed probes was also checked by the simultaneous detection with three different fluorophores (i.e., Alexa

Fluor 488-labeled EUBmix, Alexa Fluor 647-labeled TM7905, and Cy3-labeled Sacch720a, Sacch720b or Sacch933) and with Chloroflexi probes (GNSB-941 and CFX1223) and CHL1851).

7 Quantification of probe-defined bioarea was carried out as previously described

(Morgan-Sagastume et al., 2008). Briefly, each sample was diluted and homogenized before applying a very thin layer on slides. After FISH, at least 24 random fields were chosen and the relative area fluorescing with a Saccharibacteria specific probe (Cy3-labeled) and EUBmix probe (FLUOS-labeled) were estimated. The bioarea of probe-defined populations was determined by using ImageJ software

(Collins, 2007) and custom-made macros were used for post-processing and data acquisition of all images. The mean values and standard deviations from each of the measurements were calculated.

Downloaded from MAR-FISH

The combination of MAR and FISH was carried out as described by Nielsen and Nielsen (2005) and http://femsec.oxfordjournals.org/ Kindaichi et al. (2013). Slides with activated sludge (after FISH procedure) were coated with LM-1 emulsion (GE Healthcare UK Ltd., Little Chalfont, United Kingdom), exposed in the dark for 6 days and developed with a Kodak D-19 developer. Briefly, activated sludge was diluted with filtered effluent water from the same treatment plant to a final suspended solid (SS) concentration of 1 g SS L–1, and by guest on April 22, 2016 incubated in serum bottles with labeled (20 μCi) and 2 mM unlabeled substrates (except for oleic acid and ethanol, which were amended with 0.5 mM) under aerobic (3 h), anoxic (anaerobic plus 2 mM nitrate; 3 h), and anaerobic conditions for 6 h at 20°C. The bottles were sealed with gas-tight rubber stoppers. All anoxic and anaerobic preparations were carefully flushed with O2-free N2 prior to the addition of the labeled substrate. MAR-FISH results presented in this paper are shown as one series of experiments, but we conducted at least two or three independent replicates for each condition.

The organic substrates used in this study were labeled with either 3H or 14C, and covered a selection of fatty acids (acetate, propionate, butyrate, and oleic acid), sugars (glucose and

N-acetylglucosamine), amino acids (mixture from hydrolyzed 3H-Casein), pyruvate, glycerol, and ethanol. Labeled substrates were purchased from PerkinElmer, Inc. (Waltham, MA), American

Radiolabeled Chemicals, Inc. (Saint Louis, MO), or Amersham Biosciences (Little Chalfont, United

8 Kingdom).

ELF-FISH

ELF-FISH was applied to activated sludge samples from Higashihiroshima and Aalborg West WWTPs with five ELF® 97-labeled substrates (Molecular Probes, Eugene, USA); ELF® 97 esterase substrate

(ELF® 97 acetate), ELF® 97 lipase substrate (ELF® 97 palmitate), ELF® 97 β-D-galactosidase substrate

(ELF® 97 β-D-galactopyranoside), ELF® 97 β-D-glucuronidase substrate (ELF® 97 β-D-glucuronide), and ELF® 97 chitinase/N-acetylglucosaminidase substrate (ELF® 97 N-acetylglucosaminide). A Downloaded from detailed protocol containing information combining the exoenzyme approaches with FISH can be found elsewhere (Nielsen et al., 2010b). http://femsec.oxfordjournals.org/

RESULTS

Phylogenetic analysis

Nine clone libraries were constructed based on 16S rRNA genes from activated sludge obtained in one by guest on April 22, 2016 Japanese and eight Danish WWTPs. The Saccharibacteria sequences were amplified with a

Saccharibacteria specific primer set, TM7580f/Univ1492r (Hugenholtz et al., 2001; Lane, 1991). The clone libraries contained some non-saccharibacterial clones (19% of the total clones sequenced), which were excluded from the analysis. The resulting 239 non-chimeric clones could be grouped into 103

OTUs using a sequences similarity threshold of 97%. The phylogenetic relationships with closely related sequences in the database were investigated using a maximum-likelihood phylogenetic tree (Fig.

1); similar topology was seen in phylogenetic trees generated using neighbour-joining and maximum-parsimony. Using the Greengenes database (DeSantis et al., 2006), which encompasses three subdivisions, most of the OTUs obtained in this study were affiliated with subdivision 1 and 3. In addition, some OTUs constituted a separate phylogenetic clade outside of the subdivision 1 and 3 (i.e., the clade within OTU HHS-08tB03 to EBM-E10 in Fig. 1), even though the bootstrap support was only

9 26%. There is a tendency that OTUs from the 8 Danish WWTPs were affiliated with subdivision 1 and the phylogenetic clade, whereas OTUs from Japanese WWTP were affiliated with subdivision 3. All

WWTPs contained several phylotypes, indicating a high diversity of Saccharibacteria in activated sludge. Frequently detected OTUs (found in the upper part within subdivision 1 in Fig. 1) were detected in all WWTPs and closely related to the uncultured Saccharibacteria detected from activated sludge. The

16S rRNA gene from the fully sequenced genome of “Candidatus Saccharimonas aalborgensis” belonging to the subdivision 3 is closely related to the OTU AAR-B4 with 99.3% sequence similarity.

Figure 1 also shows the coverage of the probe TM7905 which targets the majority of the phylum. Downloaded from Unfortunately, the coverage of TM7305, which targets subdivision 1, cannot be evaluated due to the lack of the TM7305 binding region in the clones from this study (E. coli position, 580 to 1492). http://femsec.oxfordjournals.org/ Sequences outside subdivisions 1 and 3 have two mismatches in the probe target site of the phylum-specific TM7905 probe, except for OTUs EBM-F9 and EBM-E10.

In situ detection and abundance of probe-defined Saccharibacteria by guest on April 22, 2016 In order to detect more specifically such diverse Saccharibacteria in activated sludge, three oligonucleotide probes, Sacch720a, Sacch720b, and Sacch933, were designed (Table 2). Sacch720a and

Sacch720b probes targeted most OTUs belonging to subdivision 1 to enhance the resolution and specificity of the phylum level TM7905 probe. Sacch933 probe targeted the phylogenetic clade (i.e., the clade within OTU HHS-08tB03 to EBM-E10 in Fig. 1) to cover OTUs which could not be detected with TM7905 probe. The specificity of designed probes was further checked by hybridizing with three different fluorophores (Fig. 2). The cells that hybridized with the Sacch720a and Sacch720b probes under the optimal formamide concentration (i.e. 15%, Table 2) also hybridized with TM7905 probe and

EUBmix probe (Figs. 2A and 2B); furthermore, the cells that hybridized with Sacch933 probe in the presence of two competitor probes under the optimal formamide concentration (i.e. 20%, Table 2) also hybridized with the phylum level TM7905 probe and EUBmix probe (Figs. 2C and 2D). No

10 Sacch720a- and only some Sacch720b-positive cells were also targeted by the TM7305 probe. No hybridization signals were observed when the three probes were applied to a negative control without

Saccharibacteria. The TM7305 and TM7905 probes targeting the Saccharibacteria have previously been reported to have a low specificity (Nittami et al., 2014). The specificities of the new saccharibacterial probes (Sacch720a, Sacch720b, and Sacch933) were evaluated by the simultaneous hybridization with

Chloroflexi probes (CHL1851, GNSB-941 and CFX1223 probes) (Beer et al., 2002). The results showed that neither of the new Saccharibacterial probes also hybridized with the Chloroflexi probes

(Fig. S1). Downloaded from The abundance and morphology of Saccharibacteria in all nine WWTPs were further investigated by FISH with specific probes, including TM7905 and TM7305 probes. The total http://femsec.oxfordjournals.org/ probe-defined Saccharibacteria species (targeted by TM7905) comprised 1.3-7.3% of the total bacterial bioarea (Table 1). The abundance detected by each Saccharibacteria-specific probe varied somewhat between the different WWTPs, with relatively higher abundance observed in Higashihiroshima WWTP and lower abundances in other WWTPs (Table 1). At least three different morphotypes (thin filaments, by guest on April 22, 2016 thick filaments, and rods/cocci) were found with a variety of filaments (a range of 0.8-2.0 μm diameter) and rods or cocci (0.7-1.2 μm diameter) (Fig. 3).

Substrate uptake

The substrate uptake of probe-defined Saccharibacteria was investigated using MAR-FISH under three growth conditions (aerobic, nitrate reducing and anaerobic). Ten radiolabeled substrates were tested at the phylum level (i.e., with probe TM7905) and with Sacch933 probe using activated sludge from three

WWTPs that had a relatively high abundance of Saccharibacteria (Ejby Mølle, Marselisborg, and

Higashihiroshima). When probe-defined Saccharibacteria were partly MAR-positive (i.e. substrate uptake), the substrate uptake profiles were further investigated using group-specific probes (Table 3).

Saccharibacteria detected with the TM7905 probe took up glucose under aerobic-, anoxic-,

11 and anaerobic conditions. Filamentous Saccharibacteria from Higashihiroshima WWTP furthermore took up N-acetylglucosamine under all three growth conditions, and oleic acids and amino acids under aerobic conditions (Figs. 4A-4C). Non-filamentous Saccharibacteria from all three WWTPs took up butyrate under aerobic conditions. No Saccharibacteria took up acetate, propionate, pyruvate, glycerol, or ethanol (Table 3). Interestingly, Sacch933 probe-defined filamentous Saccharibacteria from

Higashihiroshima WWTP took up glucose and N-acetylglucosamine with similar silver grain densities under the three conditions, whereas Sacch933 probe-defined Saccharibacteria from Ejby Mølle and

Marselisborg WWTPs did not take up any substrates tested in this study (Figs. 4D-4F). TM7305 Downloaded from probe-defined filaments took up glucose with similar silver grain densities under all growth conditions, while TM7305 positive filaments from Higashihiroshima WWTP also took up oleic acid and amino http://femsec.oxfordjournals.org/ acids under aerobic conditions. Sacch720a probe-defined bacteria from Higashihiroshima WWTP did not take up any of the substrates. On the other hand, Sacch720a probe-defined non-filamentous cells from Ejby Mølle and Marselisborg WWTPs took up butyrate, while all morphotypes took up glucose

(Figs. 4G-4I). Sacch720b probe-defined cells did not take up any substrate under the conditions tested. by guest on April 22, 2016 These results clearly demonstrate a range of substrate uptake profiles within the probe-defined

Saccharibacteria with high consistency between the Japanese and Danish samples when applying broad phylum specific probes, but presence of different ecological patterns between the two Danish and the

Japanese WWTPs when applying the new and more specific probes.

Exo-enzyme activity

Nearly all TM7905 probe-defined filamentous cells from Higashihiroshima WWTP exhibited

β-galactosidase activity (Fig. 5A and 5B) and lipase activity (Table 4). In addition, most TM7305 probe-defined filamentous cells also showed lipase activity. On the other hand, TM7905 probe-defined filamentous cells from Aalborg West and Ejby Mølle WWTPs exhibited only β-galactosidase activity. In both activated sludge, other probe-defined non-filamentous Saccharibacteria (i.e. cells hybridizing with

12 TM7905 and Sacch720a probes) did not exhibit any exo-enzyme activities or were not sufficiently active to be detected (Table 4). No activities were observed for esterase (Fig. 5C and 5D),

β-glucuronidase, Chitinase/N-acetylglucosaminidase with all probes tested (Table 4). Activity assays for each ELF-substrate were confirmed by the presence of ELF-positive signals among other

EUB-FISH-positive cells in the sludges.

Potential enzymes responsible for the observed exo-enzymatic reactions were identified by searching for pfams and export signal peptides in the four genomes available for Saccharibacteria obtained from wastewater (Albertsen et al., 2013). All enzymatic reactions annotated within the enzyme Downloaded from classifications: Esterase, β-galactosidase, lipase (palmitate), glucoronidase, chitinase and

N-acetylglucosaminidase are listed in Table S1. To further investigate the potential for extracellular http://femsec.oxfordjournals.org/ degradation of sugar compounds we identified putative extracellular enzymes in Candidatus

Saccharimonas aalborgensis (CP005957) using PSORTb 3.0 (Yu et al., 2010). In total 11 proteins were predicted as extracellular with one annotated as a putative polysaccharide deacetylase (YP_007985614).

by guest on April 22, 2016 DISCUSSION

The present study provides new insights on the abundance, phylogenetic diversity and ecophysiology of uncultured bacteria belonging to the candidate phylum Saccharibacteria in nine full-scale WWTPs from two continents. The abundance and consistent distribution of Saccharibacteria in this and previous studies (e.g. Mielczarek et al., 2012) support that Saccharibacteria is an important group of organisms for the activated sludge process. The presence of 103 phylotypes in the examined plants indicates high diversity in this phylum. In addition, the phylogenetic analysis revealed the presence of a hitherto undescribed clade outside the well-described subdivisions 1 and 3. This clade is possibly belonging to the subdivision 2 (Hugenholtz et al., 2001; Ferrari et al. 2014). Although several genomes of

Saccharibacteria are available to predict their carbon metabolism, which predicts fermentation of sugars and oxygen tolerance (Albertsen et al., 2013; Kantor et al., 2013), new group-specific probes in the

13 present study enabled detailed in situ ecophysiological studies, which revealed that the probe-defined

Saccharibacteria primarily took up monomeric glucose (and glucosamine) under all tested growth conditions (aerobic-, nitrate reducing- and anaerobic conditions). Furthermore, probe-defined

Saccharibacteria also exhibited uptake of butyrate, oleic acid, and amino acids unexpected from the available genomes so far. This inconsistency is most likely due to the available genomes only representing subdivision 3. Complete genomes of Saccharibacteria within subdivision 1, which is the most abundant phylotype in the examined activated sludges, are needed to clarify the versatile carbon metabolism and in situ ecophysiology of Saccharibacteria in activated sludge. Downloaded from Silver grain densities of MAR analysis were similar under the three electron acceptor conditions conditions. A variable yield under such conditions would have been expected for organisms http://femsec.oxfordjournals.org/ dependent on oxidative phosphorylation. We therefore hypothesize that Saccharibacteria lack capabilities of oxidative phosphorylation. The restricted uptake pattern is supported by previous

MAR-FISH and stable isotopic probing (SIP) studies (Thomsen et al., 2002; Ariesyady et al., 2007;

Nielsen et al., 2012b) and by the absence of genes coding for the electron transport chain, the by guest on April 22, 2016 tricarboxylic acid (TCA) cycle, the Entner–Doudoroff (ED) pathway, the Embden–Meyerhof–Parnas

(EMP) pathway, denitrification, and nitrification in the presently available Saccharibacteria genomes obtained from activated sludge (Albertsen et al., 2013) and an acetate amended aquifer (Kantor et al.,

2013). Hence, the central metabolism of Saccharibacteria can be hypothesized to encompass an obligate fermentative metabolism of glucose which is independent on the presence of oxygen. Some

Saccharibacteria show more diverse substrate preferences across sludges from different WWTPs and thus indicate metabolic diversity within the phylum. The fermentative metabolism indicates niche differentiation between the filamentous members of the phylum Saccharibacteria and the more abundant

Chloroflexi in activated sludge. Chloroflexi filaments also utilize sugar compounds, but are primarily active under aerobic condition and appear unable to ferment (except for pure cultures) under in situ conditions (Kragelund et al., 2007; Nielsen et al., 2009).

14 Some filamentous Saccharibacteria also utilized the glucose derivative N-acetylglucosamine under aerobic conditions, which constitute a substantial part of the cell-wall peptidoglycan and lipopolysaccharides (Barker and Stuckey, 1999). Peptidoglycan depolymerization (i.e., cell decay) leads to liberation of N-acetylglucosamine to the bulk liquid, which could provide a substrate for

Saccharibacteria and explain the wide distribution in many environments. The MAR experiments also indicated that filamentous Saccharibacteria from the Higashihiroshima WWTP were able to take up fatty acids (oleic acid), which was confirmed by the presence of lipase activity from the ELF assay.

However, no uptake of fatty acid nor presence of extracellular lipase activity was not seen in the Danish Downloaded from plants and might therefore indicate different ecophysiological populations, although this could not be reflected in the phylogenetic analysis of the 16S rRNA gene sequences. http://femsec.oxfordjournals.org/ The specificities of the new designed probes were confirmed in silico and by double hybridization together with the phylum level TM7905 probe due to the lack of proper isolates. The probes Sacch720a and Sacch720b provide increased resolution within subdivision 1 and interestingly targets two morphologically distinct filaments. Non-filamentous morphotypes was observed with by guest on April 22, 2016 double hybridization using the Sacch720a and TM7905 probes in most WWTPs. This is consistent with previous observations using the TM7905 and TM7305 probes, where non-filamentous morphotypes were detected in the subdivision 1 (Thomsen et al., 2002). These results suggest that non-filamentous morphotypes are widespread throughout the phylum Saccharibacteria. However, morphological characteristics of Saccharibacteria in activated sludge should be confirmed under in situ conditions as pure cultures can bias the morphological characteristics (Soro et al., 2014). The Sacch933 probe was originally designed for the detection of a group of Saccharibacteria that was not covered with the phylum specific probe TM7905. However, our results showed that most cells hybridizing with

Sacch933 also hybridized with the phylum level probe TM7905 (Fig. 2), even though the target sequences have two mismatches against the probe TM7905, except for OTU EBM-F9 could be detected with both Sacch933 and TM7905 probes in silico (Fig. 1). Therefore, it is possible that more

15 stringent hybridization conditions are required for enhanced specificity of TM7905 and support previous reports on the lack of specificity of this probe (Nittami et al., 2014; Wright et al. 2014). The specificity of the Sacch933 probe could be enhanced when applied together with two competitor probes. There is a limitation of Sacch933 probe even with the two competitor probes; the Sacch933 probe possibly detects some member of Candidate division OD1 (Parcubacteria) based on the TestProbe analysis (Quast et al.,

2013). However, the members of the Candidate division OD1 does not appear to be abundant in activated sludge as determined from amplicon sequencing (Nielsen JL, unpublished; Saunders et al.,

2015). The moderate specificity of the TM7905 and TM7305 probes previously reported (Nittami et al., Downloaded from 2014; Wright et al. 2014), were also evaluated for the new saccharibacterial Sacch720a and Sacch720b probes by double hybridization with Chloroflexi specific probes (i.e., GNSB-941, CFX1223, http://femsec.oxfordjournals.org/ CHL1851). In the nine activated sludge samples investigated no cells were positive with both the

Chloroflexi probes and either of the new probes. Therefore, we hypothesize that the Sacch720a and

Sacch720b has high specificity and the obtained in situ physiologies indeed represents saccharibacterial physiology in activated sludge. by guest on April 22, 2016 The fact that at least three different morphotypes could be detected with the newly designed probes supports the presence of a variety of morphologies within Saccharibacteria. On the other hand, an isolated Saccharibacteria species (TM7 isolate UB2523) exhibited a pleomorphic characteristic when cultivated in dual-species biofilms (Soro et al., 2014). In natural environments including activated sludge processes, Saccharibacteria are present in the complex microbial communities and thus the change of morphology of identical species might occur. Further studies on the in situ morphology of

Saccharibacteria are therefore needed to clarify the morphological diversity of Saccharibacteria in the environments.

In conclusion, the detailed phylogeny and ecophysiology of the candidate phylum

Saccharibacteria in activated sludge were investigated using the full-cycle rRNA approach and MAR- and ELF-FISH analyses. Saccharibacteria from nine WWTPs were phylogenetically diverse and

16 include a clade outside subdivisions 1 and 3. Based on the detailed phylogenetic tree of Saccharibacteria, we designed new group-specific probes in the phylum Saccharibacteria. MAR- FISH analyses indicated a more versatile carbon metabolism such as utilization of butyrate, oleic acid, and amino acids which was not predicted from available genomes. To better understand the phylogenetic and physiological diversity of Saccharibacteria there is a need for obtaining complete reference genomes that cover the genetic diversity. These can provide grounds for initial hypothesis on the metabolic capability and insight into the enzymes responsible for their ecophysiology, and be verified or rejected by a combination of probes and in situ techniques like the ones applied in this study. Downloaded from

ACKNOWLEDGEMENTS http://femsec.oxfordjournals.org/ This work was carried out at the Analysis Center of Life Science and the Radioisotope Research Center,

Hiroshima University and at the Department of Chemistry and Bioscience, Aalborg University.

FUNDING by guest on April 22, 2016 This work was supported by the JSPS KAKENHI [Grant Number 26650145] and the Danish Research

Council for Strategic Research via the Centre “EcoDesign” [Grant Number 09-067230]. T.K. was supported by The Institutional Program for Young Researcher Overseas Visits [Strategic Fostering

Program for Young Researchers Engaged in Natural Sciences toward the Establishment of the

Sustainable Society] from the Japan Society for the Promotion of Science (JSPS). The authors declare no conflict of interest.

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21 Downloaded from http://femsec.oxfordjournals.org/ by guest on April 22, 2016

Figure 1 Maximum-likelihood phylogenetic tree of Saccharibacteria-related OTUs obtained from nine activated sludge clone libraries. Colored OTUs represent OTUs obtained from different WWTPs: HHS (Higashihiroshima), AAW (Aalborg

West), SDB (Sønderborg), RDS (Randers), VIB (Viborg), EBM (Ejby Mølle), HOR (Horsens), AAR (Aars), and MSB

(Marselisborg). Sequences retrieved in the present study are colored. The numbers in parentheses indicate the frequencies of the identical clones analyzed from each library. The scale bar represents the number of nucleotide

22 changes per position. Open circles at the nodes represent bootstrap values >90% support obtained from 1000 resamplings. Twenty Thermotoga sequences were used as outgroup. The gray boxes represent the subdivision of

Saccharibacteria based on the Greengenes taxonomy. The vertical colored lines for each probe indicate the OTUs possibly hybridized with the probe with zeromismatch. Downloaded from http://femsec.oxfordjournals.org/ by guest on April 22, 2016

Figure 2 FISH micrographs of Saccharibacteria in Ejby Mølle WWTP activated sludge (A and D), Higashihiroshima

WWTP activated sludge (B), and Marselisborg WWTP activated sludge (C). FISH were performed with three different

fluorophores, the Alexa Fluor 488-labeled EUBmix probes (green), Alexa Fluor 647-labeled probe TM7905 (blue),

and Cy3-labeled probe Sacch720a with a competitor probe (red) (A), Cy3-labeled probe Sacch720b with a

23 competitor probe (red) (B), or Cy3-labeled probe Sacch933 with two competitor probes (red) (C and D). The scale

bars represent 10 μm. Sacch720a, Sacch720b, and Sacch933 probe-hybridized cells appear white (green + red +

blue) in all panels. Other Sacchribacterial cells appear cyan (green + blue) and other bacterial cells appear green in

all panels. Arrows indicate as follows: a, 720a probehybridized filamentous Saccharibacteria; b, 720a

probe-hybridized cocci-shaped Saccharibacteria; c, 720b probe-hybridized filamentous Saccharibacteria; d,

Sacch933 probe-hybridized filamentous Saccharibacteria; e, other filamentous bacteria; f, filamentous

Saccharibacteria; g, rods/cocci-shaped Saccharibacteria.

Downloaded from http://femsec.oxfordjournals.org/ by guest on April 22, 2016

Figure 3 Morphotypes of Saccharibacteria detected by using Saccharibacteria specific probes and typical FISH images. In situ hybridizations were performed with FLUOS-labeled EUBmix probes (green) and Cy3-labeled Saccharibacteria specific probes (red). Saccharibacteria-related cells appear yellow and other bacteria appear green. The scale bar represents 10 μm.

24 Downloaded from http://femsec.oxfordjournals.org/ by guest on April 22, 2016

Figure 4 MAR-FISH micrographs of Higashihiroshima WWTP activated sludge incubated with labeled oleic acid (A-C),

Ejby Mølle WWTP activated sludge incubated with labeled N-acetylglucosamine (D-F), and Marselisborg WWTP activated sludge incubated with labeled glucose (G-I). In situ hybridizations were performed with a combination of the

FLUOS-labeled EUBmix probes (green) and Cy3-labeled probe TM7905 (A), Cy3-labeled probe Sacch933 (D), or

Cy3-labeled probe Sacch720a (G). Saccharibacteria-related cells (A, D, and G) appear yellow and other bacteria appear green. Panel B, E, and H are bright-field MAR images, corresponding to the FISH images (A, D, and G, respectively).

Panel C, F, and I are overlay images showing that Saccharibacteria took up oleic acid (C), did not take up

25 N-acetylglucosamine (F), but took up glucose (I). The scale bars represent 10 μm. Arrows indicate MAR-positive or

MAR-negative cells.

Downloaded from http://femsec.oxfordjournals.org/

Figure 5 ELF-FISH micrographs of Higashihiroshima WWTP activated sludge incubated with ELF® 97 β-Dgalactosidase by guest on April 22, 2016 substrate (A and B) and ELF® 97 esterase substrate (C and D). In situ hybridizations were performed with a combination of the FLUOS-labeled EUBmix probes (green) and Cy3-labeled probe TM7905 (A and C). Saccharibacteria-related cells

(A and C) appear yellow, other bacteria appear green. Panel B and D are ELF images, corresponding to the FISH images, showing galactosidase activity (white signals) (B) and no esterase activity (D). The scale bars represent 10 μm.

26 Table 1. Abundance of probe-defined groups of Saccharibacteria in full-scale WWTPs with different configurations.

Population Abundance of probe-defined Saccharibacteria3 WWTPs1 Configration2 equivalent TM7905 TM7305 Sacch933 Sacch720a Sacch720b

Higashihiroshima (M) C, N, DN, EBPR 50,000 7.3±2.5 2.7±0.9 0.8±0.5 1.0±0.2 1.2±0.3 Aalborg West (M) C, N, DN, EBPR, CP 220,000 3.3±1.1 2.0±0.4 0.5±0.2 0.9±0.3 0.6±0.2 Sønderborg (M) C, N, DN, EBPR, CP 28,000 1.8±0.5 1.2±0.4 0.4±0.1 0.9±0.1 0.5±0.1 Randers (M) C, N, DN, EBPR 75,000 2.6±1.3 1.7±0.7 0.5±0.2 1.2±0.2 0.3±0.1 Viborg (M) C, N, DN 68,000 1.3±0.4 0.3±0.1 0.4±0.1 0.8±0.3 0.4±0.1 Ejby Mølle (M/I) C, N, DN, EBPR 236,000 3.3±0.9 1.5±0.4 0.7±0.2 0.4±0.2 0.5±0.1 Horsens (M) C, N, DN 140,000 2.3±0.7 0.7±0.3 0.4±0.2 0.6±0.3 <0.3 Aars (I) C, N, DN 60,000 5.4±1.5 1.7±0.6 0.7±0.4 1.3±0.5 0.4±0.2 Marselisborg (M/I) C, N, DN 157,000 4.5±1.2 1.6±0.4 0.4±0.1 0.9±0.2 <0.3

1 M and I represent primarily municipal and industrial wastewater, respectively. Downloaded from

2 C, carbon removal; N, nitrification; DN, denitrification; EBPR, enhanced biological phosphorus

removal; CP, chemical phosphorus removal. http://femsec.oxfordjournals.org/

3 Numbers are given as percentage relative to EUBmix.

Table 2. 16S rRNA-targeted oligonucleotide probes used for detection of Saccharibacteria in WWTPs.

1

Probe Sequence (5’–3’) FA (%) Specificity Reference by guest on April 22, 2016 EUB338 GCT GCC TCC CGT AGG AGT 0-50 Most Bacteria Amann et al., 1990 EUB338 II GCA GCC ACC CGT AGG TGT 0-50 Planctomycetales Daims et al., 1999 EUB338 III GCT GCC ACC CGT AGG TGT 0-50 Verrucomicrobiales Daims et al., 1999 TM7905 CCG TCA ATT CCT TTA TGT TTT A 20 Candidate division TM7 Hugenholtz et al., 2001 TM7305 GTC CCA GTC TGG CTG ATC 30 Subdivision 1 of candidate Hugenholtz et al., 2001 division TM7 Sacch933 CAC GCT CCA CCA CTT GTG 20 Some members of This study Saccharibacteria Comp1 Sacch9332 CAC GCT CCA CCG CTT GTG − Competitor for Sacch933 This study Comp2 Sacch9332 CAT GCT CCA CCA CTT GTG − Competitor for Sacch933 This study Sacch720a3 TTA CCT GCC TAC GCC ATC 15 Some members of This study Saccharibacteria Sacch720b4 TTA TCT GCC TAC GCC ATC 15 Some members of This study Saccharibacteria GNSB-941 AAA CCA CAC GCT CCG CT 35 Phylum Chloroflexi Gich et al., 2001 CFX1223 CCA TTG TAG CGT GTG TGT MG 35 Phylum Chloroflexi Bjornsson et al., 2002 CHL1851 AAT TCC ACG AAC CTC TGC CA 35 Eikelboom morphotype 1851 Beer et al., 2002 1 Formamide concentration in the hybridization buffer (v/v).

2 Used as unlabeled competitor probes together with probe Sacch933.

3 Unlabeled probe Sacch720b was used as the competitor to enhance the specificity.

4 Unlabeled probe Sacch720a was used as the competitor to enhance the specificity.

27 Table 3. Substrate uptake profiles of probe-defined Saccharibacteria determined by MAR-FISH. Higashihiroshima Ejby Mølle Marselisborg

2

Probe Substrate Aerobic Anoxic Anaerobic Aerobic Anoxic Anaerobic Aerobic Anoxic Anaerobic TM7905 Acetate −/−1 −/− −/− −/− −/− −/− −/− −/− −/− Propionate −/− −/− −/− −/− −/− −/− −/− −/− −/− Butyrate −/+ −/− −/− −/+ −/− −/− −/+ −/− −/− Pyruvate −/− −/− −/− −/− −/− −/− −/− −/− −/− Glycerol −/− −/− −/− −/− −/− −/− −/− −/− −/− Oleic acid +/− −/− −/− −/− −/− −/− −/− −/− −/− Glucose +/+ +/+ +/+ +/+ +/+ +/+ +/+ +/+ +/+ N-acetylglucosamine +/− +/− +/− −/− −/− −/− −/− −/− −/− Downloaded from Amino acids +/− −/− −/− −/− −/− −/− −/− −/− −/− Ethanol −/− −/− −/− −/− −/− −/− −/− −/− −/−

Sacch933 Acetate − − − − − − − − −

Propionate − − − − − − − − − http://femsec.oxfordjournals.org/ Butyrate − − − − − − − − − Pyruvate − − − − − − − − − Glycerol − − − − − − − − − Oleic acid − − − − − − − − − Glucose + + + − − − − − − N-acetylglucosamine + + + − − − − − − Amino acids − − − − − − − − − Ethanol − − − − − − − − − by guest on April 22, 2016

TM7305 Butyrate − ND3 ND − ND ND − ND ND Oleic acid + − − − ND ND − ND ND Glucose + + + + + + + + + N-acetylglucosamine − − − − ND ND − ND ND Amino acids + − − − ND ND − ND ND

Sacch720a Butyrate −/− ND ND −/+ −/− −/− −/+ −/− −/− Oleic acid −/− −/− −/− −/− ND ND −/− ND ND Glucose −/− −/− −/− +/+ −/+ −/+ +/+ +/+ +/+ N-acetylglucosamine −/− −/− −/− −/− ND ND −/− ND ND Amino acids −/− −/− −/− −/− ND ND −/− ND ND

Sacch720b Butyrate ND ND ND − ND ND − ND ND Oleic acid − ND ND ND ND ND ND ND ND Glucose − − − − − − − − − N-acetylglucosamine − ND ND ND ND ND ND ND ND Amino acids − ND ND ND ND ND ND ND ND 1 Filamentous/non-filamentous cells; +, MAR-positive; −, MAR-negative.

2 Refers to anaerobic conditions in the presence of 2 mM nitrate.

28 3 Not determined.

Table 4. Exoenzyme activity of probe-defined Saccharibacteria determined by ELF-FISH. ELF substrate TM7905 TM7305 Sacch933 Sacch720a Sacch720b Esterase −/−1 − − ND3 ND β-D-glucuronidase −/− − − ND ND β-D-galactosidase +/− − − −/− − Lipase +2/− +2 − −/− − Chitinase/ −/− − − ND ND N-acetylglucosaminidase 1 Filamentous/non-filamentous cells; +, ELF-positive; −, ELF-negative.

2 Only found in Higashihiroshima WWTP. Downloaded from

3 Not determined.

http://femsec.oxfordjournals.org/ by guest on April 22, 2016

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