Culture-Independent Analyses Reveal Novel Anaerolineaceae as Abundant Primary Fermenters in Anaerobic Digesters Treating Waste Activated Sludge

Anaerobic digestion for biogas production is reliant on the tightly coupled synergistic activities of complex microbial consortia. Members of the uncultured A6 phylotype, within the phylum Chloroflexi, are among the most abundant genus-level-taxa of mesophilic anaerobic digester systems treating primary and surplus sludge from wastewater treatment plants, yet are known only by their 16S rRNA gene sequence. This study applied metagenomics to obtain a complete circular genome (2.57 Mbp) from a representative of the A6 taxon. Preliminary annotation of the genome indicates these organisms to be anaerobic chemoorganoheterotrophs with a fermentative metabolism. Given their observed abundance, they are likely important primary fermenters in digester systems. Application of fluorescence in situ hybridisation probes designed in this study revealed their morphology to be short filaments present within the flocs. The A6 were sometimes co-located with the filamentous Archaea Methanosaeta spp. suggesting potential undetermined synergistic relationships. Based on its genome sequence and morphology we propose the species name Brevefilum fermentans gen. nov. sp. nov.


INTRODUCTION
Anaerobic digestion (AD) involves the conversion of organics to valuable methane, which is facilitated by the tightly coupled synergistic activities of complex microbial communities. The process essentially consists of four sequential microbial-mediated processes: hydrolysis, fermentation (acidogenesis), acetogenesis (dehydrogenation) and methanogenesis (acetoclastic or hydrogenotrophic) (Vanwonterghem et al., 2014). Members of the phylum Chloroflexi are widespread in full-scale ADs, constituting up to 50% of the bacterial community, and are largely confined to the family Anaerolineaceae (Nelson et al., 2011;Kirkegaard et al., 2017). Surprisingly, although their abundance indicates they must play a considerable role in these systems, their physiology and ecology is largely unknown. Most species of the Anaerolineaceae were isolated from anaerobic digester systems and have a fermentative metabolism, utilizing carbohydrates and proteinaceous carbon sources under anaerobic conditions (Sekiguchi et al., 2003;Yamada et al., 2006Yamada et al., , 2007Sun et al., 2016). A role in fermentation in AD systems is additionally supported by the annotation of available genomes derived from metagenomes (Xia et al., 2016) and with in situ evidence for the Chloroflexi phylum (Ariesyady et al., 2007). An in-depth understanding of the ecology and function of the Chloroflexi in biogas systems requires the characterisation of the abundant genera of the phylum.
A recent large scale amplicon sequencing survey of Danish full-scale AD communities revealed the A6 phylotype, a member of the Anaerolineaceae known only by their 16S rRNA gene sequence, to be among the most abundant genus-level-taxa in these systems; at times being present in excess of 20% of the amplicon reads . Advances in sequencing and metagenomic analyses enable the attainment of full genomes from the uncultured majority of microorganisms (Wrighton et al., 2012;Albertsen et al., 2013). In the absence of a pure culture, the aim of this study was to apply metagenomics to obtain a genome from a representative of the A6 phylotype, giving the first insight into their physiology.

Metagenome Preparation and Genome Binning
Metagenome sequencing and genome binning was performed essentially as detailed by Kirkegaard et al. (2016). Metagenomes were prepared from sludge obtained from the anaerobic digester tank at Fredericia wastewater treatment plant in Denmark. The Fredericia plant has 2 × 2000 m 3 mesophilic digester tanks with Cambi TM thermal high-pressure (THP) pre-treatment of influent sludge and treats 8000 metric tonnes dry sludge per year. Sludge was obtained from the digester at two different time points 36 weeks apart (March and December, 2011) enabling later differential coverage binning of genomes (Albertsen et al., 2013). The DNA was extracted from AD sludge using the FastDNA R Spin kit for soil (MP Biomedicals, Santa Ana, CA, United States) following the standard protocol with minor modifications, as recommended by Albertsen et al. (2015). These differences included a four times increase in the duration of bead-beating and a sludge input volume of 50 µl.
Illumina TruSeq PCR free libraries and Nextera mate pair libraries were prepared according to the manufacturers' protocol and paired-end sequenced (2 × 150 bp) on the Illumina HiSeq 2000 platform. The metagenomic reads were co-assembled using default settings in CLC Genomics Workbench (CLC Bio v. 7.5.1., Aarhus, Denmark). Reads were mapped to the assembly separately for each sample using default settings in CLC Genomics Workbench. The assembly and mapping information was exported as .fasta and .csv files, respectively, which were processed with the mmgenome workflow script 'data.generation.2.1.0.sh' to generate the files necessary for the binning process. Binning was carried out in the R environment using the mmgenome package 1 . The raw metagenome reads and the annotated genome sequence data 1 https://github.com/MadsAlbertsen/mmgenome  Kirkegaard et al. (2017) which the reader is referred to for further details.
have been submitted to the European Nucleotide Archive (ENA) under the study accession number PRJEB19949.

Genome Annotation
Genome annotation was performed in the 'MicroScope' annotation pipeline (Vallenet et al., 2013). Automatic annotations were validated manually for the genes involved in metabolic pathways of interest with the assistance of the integrated MicroCyc (Caspi et al., 2014) and KEGG (Kyoto Encyclopedia of Genes and Genomes) (Kanehisa et al., 2014) (Quast et al., 2013). Additional sequences were aligned with the online SINA aligner with default settings (Pruesse et al., 2012) and imported into ARB. The alignment was trimmed and variable regions removed using a custom 40% base frequency filter giving 1372 aligned positions for tree calculation. Herpetosiphon aurantiacus was used to root the tree. Bootstrap values from 100 re-samplings are indicated for branches when >50%: white dots, >50%; gray, >70%; black, >90%. The scale bar represents substitutions per nucleotide base.
FIGURE 3 | Cell envelope classification of CAMBI-1. Analysis was based on a search of the genome for genes encoding PFAM proteins (Finn et al., 2016) that are specific to archetypical mono-(M) or diderm bacteria with lipopolysaccharides (DL) or atypical diderm bacteria (DA) (as detailed previously by Albertsen et al., 2013). These include proteins involved in lipopolysaccharide synthesis (LPS), outer membrane associated proteins (OMP), and proteins associated with septum formation and sporulation. The percentage prevalence of each PFAM is given for each listed phylum. Phyla included are represented by all complete genomes (at least four each) in the IMG database (release 3.5) (Chen et al., 2017). The numbers shown in the column for CAMBI-1 represent the number of hits for a given PFAM protein in the analyzed genome. The PFAM profile of CAMBI-1 is similar to those of archetypical monoderm bacteria, including other members of the Chloroflexi.

Probe Design and Fluorescence In Situ Hybridization (FISH)
Phylogenetic analysis and probe design was performed with the ARB software (Ludwig et al., 2004). Potential probes were assessed in silico with the mathFISH software (Yilmaz et al., 2011). The Ribosomal Database Project (RDP) PROBE MATCH function was used to screen for non-target sequences with single base indels (McIlroy et al., 2011;Cole et al., 2014). In order to improve the fluorescence in situ hybridization (FISH) signal to background ratio, both the 5 and 3 ends of oligonucleotide FISH probes were labeled with either the sulfoindocyanine dyes Cy3 or Cy5, or the 5(6)-carboxyfluorescein-N-hydroxysuccinimide ester (FLUOS) [DOPE-FISH (Stoecker et al., 2010)]. Probe validation and optimisation was based on generated formamide dissociation curves (Daims et al., 2005). With no pure cultures available, both probes were optimized using biomass with high abundance of the target organism from the anaerobic digester located at Ejby Mølle, Denmark (sampled August, 2013). In addition, CFX-A6-1278 was validated against Pelolinea submarina MO-CFX1 T which has a single base mismatch. Pure cultures with a single base mismatch to the CFX-A6-450 probe were not available. Probes designed in this study were deposited into the probeBase database (Greuter et al., 2016). The ARCH915 and the MX825mix (MX825; MX825b; MX825c) (Raskin et al., 1994;Crocetti et al., 2006) probes were applied to target the domain Archaea and the genus Methanosaeta, respectively. Quantitative FISH (qFISH) values were calculated as a percentage area of the total biovolume, stained with the 4 ,6-diamidino-2-phenylindole (DAPI) DNA stain (50 µM, 1 h, at 4 • C), which hybridized with the specific probe. The qFISH analyses were based on 25 fields of view taken at 630× magnification using the Daime image analyses software (DOME, Vienna, Austria) (Daims et al., 2006). Microscopy was performed with either an Axioskop epifluorescence microscope (Carl Zeiss, Oberkochen, Germany) or a White Light Laser Confocal Microscope (Leica TCS SP8 X) fitted with a 405 nm diode laser (Leica Microsystems, Kista, Sweden).

RESULTS AND DISCUSSION
Amplicon sequencing survey data of full-scale ADs at wastewater treatment plants in Denmark showed a high abundance of the A6 phylotype in many of the mesophilic anaerobic digester tanks,   , 2017). There were no non-target hits for either probe. * * Optimal hybridisation formamide concentration % [v/v]. * * * The CFX-A6-1278 probe did not give a positive signal when applied to Pelolinea submarina MO-CFX1 T (at the recommended formamide concentration), which represents the non-target single base mismatched sequences covered by this competitor probe -the CFX-A6-1278_C2 probe is therefore not required. * * * * Addition of these helper probes did not improve fluorescence when applied with their respective probes and are therefore not required.

Measurement AD location Ejby Mølle Slagelse Randers Aalborg East
Archaea % biovolume * 4 ± 2 2 ± 1 5 ± 1 2 ± 1 Values are averages ± standard deviation, calculated from * 25 image sets or * * 3 manual counts of 100 randomly selected Methanosaeta filaments (>10 µm in length). Methanosaeta filaments were considered positive for association with A6 filaments if multiple of the latter appeared horizontally attached to the former. 12 other mesophilic ADs located in Denmark were screened but excluded from analyses due to high background fluorescence or the absence or low abundance of the A6.
but not in the primary or secondary sludge fed into these systems, suggesting that they are growing and well-adapted to mesophilic digester environment (Figure 1). In order to obtain genomes for the A6 taxon, metagenomes were generated for the Fredericia AD plant due to the observed high abundance of the target phylotype (representing up to 10% of the metagenome reads). A complete circular genome (CAMBI-1), classified to the novel MiDAS taxonomy defined A6 genus  based on its 16S rRNA gene sequence, was successfully assembled from the metagenomes (see Table 1 for details). Phylogenetic analysis of the 16S rRNA gene revealed that CAMBI-1 clusters together with isolates of the Anaerolineaceae, sharing 85-90% 16S rRNA gene sequence identity (Figure 2). Based on the recommendations of Yarza et al. (2014), this indicates that CAMBI-1 should be considered to represent a novel genus within the family. Examination of the CAMBI-1 genome for PFAM proteins related to archetypic mono-and diderm cell envelopes, revealed a monoderm cell envelope architecture consistent with other Chloroflexi (Figure 3). The genome annotation and specialized searches using the PilFind program (Imam et al., 2011) did not reveal any genes associated with flagella, fimbriae or pili, suggesting a non-motile lifestyle. Putative genes associated with spore coat polysaccharide biosynthesis protein SpsC (CFX1CAM_0088; 1106) were annotated (Cangiano et al., 2014), although definitive candidates for other spore related genes were not found and their ability to form spore like structures is unclear.
The CAMBI-1 genome lacks a cytochrome oxidase, electron transport chain complexes and several key enzymes required for a complete TCA cycle, indicating a strict anaerobic metabolism. Annotation of a catalase (CFX1CAM_0578) and superoxide dismutase (CFX1CAM_2274) indicates some resistance to oxidative stress. Genes for the dissimilarity reduction of sulfate, nitrate or nitrite were also not annotated. Although an ability for denitrification was not annotated, the organism possesses a putative nitric oxide reductase (norV) (CFX1CAM_0414) and a putative hydroxylamine reductase (hcp) (CFX1CAM_0418), which both have suggested involvement in protection against nitrosative stress (Vine and Cole, 2011). Key genes for the Wood-Ljungdahl pathway and the Calvin-Benson-Bassham cycle were not annotated, indicating an inability to fix carbon for autotrophy. Potential for the pentose phosphate and Embden-Meyerhof-Parnas glycolysis pathways were present. Several annotated genes suggest a fermentative physiology consistent with other members of the family Anaerolineaceae (Figure 4 and Table 2). Pyruvate can be converted to acetyl-CoA by a pyruvate: ferredoxin oxidoreductase (CFX1CAM_0326), pyruvate dehydrogenase (CFX1CAM_1724-1726) or a pyruvate formate lyase (CFX1CAM_0333), with formate released from activity of the latter potentially oxidized to CO 2 by an annotated formate dehydrogenase (CFX1CAM_1212). Potential fermentation by-products from acetyl-CoA include acetate, mediated by an acetyl-CoA synthetase (CFX1CAM_0825; 1292), and ethanol, facilitated by acetaldehyde (CFX1CAM_1715) and alcohol dehydrogenases (CFX1CAM_0055). The annotation of putative genes associated with the methylmalonyl-CoA pathway (CFX1CAM_1019; 1020; 2064-2067) indicates that propionate could be produced as a metabolic by-product from the fermentation of amino acids. Annotated tungsten-containing aldehyde ferredoxin oxidoreductases (AORs) (CFX1CAM_1238; 2051) may function to oxidize aldehydes derived from amino acid oxidation (Heider et al., 1995). Several described members of the Anaerolineaceae ( Table 2) produce hydrogen as a fermentation by-product. However, definitive evidence for a hydrogenase was not found in the CAMBI-1 genome.
Fluorescence in situ hybridisation probes were designed to visualize the morphology of the A6 in situ ( Table 3). The CFX-A6-450 and CFX-A6-1278 probes were designed to cover the phylotype. These can be applied together with different fluorochromes, where the overlap gives a higher confidence in specificity, or with the same fluorochrome to give a higher coverage of the group and to increase the signal to background ratio that can be problematic with AD samples. Application of these probes to the Fredericia AD biomass, and several additional full-scale digesters, revealed that these organisms form short filaments that are typically approximately 0.3 µm thick and 5-10 µm long ( Figure 5C), but were occasionally observed at lengths of >100 µm. Unlabelled helper probes were designed for the CFX-A6-1278 and CFX-A6-450 probes, but only CFX-A6-1278_H1 gave an increase in fluorescence and is recommended for use (Table 3). Competitor probes were designed to cover un-validated single base mismatches in non-target sequences ( Table 3). Stringency of the CFX-A6-1278 probe was supported by its application to P. submarina MO-CFX1 T -a non-target isolate with a single mismatch to the probe -which gave no positive fluorescent signal. Unlike some prominent wastewater-related Chloroflexi (Kragelund et al., 2007(Kragelund et al., , 2011Speirs et al., 2009), the A6 are covered by the EUBmix FISH probe set routinely applied to cover most members of the domain bacteria (Amann et al., 1990;Daims et al., 1999).
The A6 were distributed throughout the flocs and were sometimes observed co-located with the filamentous Archaea Methanosaeta spp. (Figure 5), indicating the potential for synergistic relationships. Parallel growth of these two filamentous genera was observed in multiple plants but was only frequent in the Ejby Mølle AD (23% of Methanosaeta filaments having associated A6; see Table 4). Unfortunately, the low FISH signal to background ratio for the thin A6 filaments did not permit qFISH studies or statistical co-location analyses (Daims et al., 2006); noting that visual assessment often indicated a higher abundance of A6 relative to the Methanosaeta spp., with most of both genera growing independent of contact with the other. Amplicon sequencing surveys indicate that the Methanosaeta are the most abundant methanogenic archaeal genus in mesophilic ADs located at WWTPs in Denmark , which was supported by qFISH in this study where they made up approximately half of the archaeal biovolume ( Table 4). As such, the potential synergistic relationship with the A6 filaments may have important implications for methane production in some full-scale AD systems (e.g., Ejby Mølle). Methanosaeta are widely known to be acetoclastic methanogens (Whitman et al., 2014), which would therefore benefit from the use of any acetate theoretically produced by the fermentative A6. It has also been demonstrated that some Methanosaeta species in ADs can utilize electrons sourced through direct interspecies electron transfer (DIET), from ethanol oxidizing Geobacter spp., to reduce CO 2 to methane . Thus, the A6 may transfer excess electrons from the oxidation of organic carbon to the Methanosaeta via DIET, facilitated by the observed close proximity of the two organisms. It is thought that cytochromes and electrically conductive pili structures facilitate electrons flow to the recipient organism . A single membrane associated polyheme cytochrome c (CFX1CAM_1800) was annotated which had low homology to known DIET cytochromes (22% amino acid sequence similarity with OmcS from Geobacter sulfurreducens (Acc. No. AAR35877)), though no pili associated genes were identified in the CAMBI-1 genome. It may be that novel conductive structures are utilized by these organisms, or the CAMBI-1 genome may not represent the A6 species co-located with the Methanosaeta. Further work into the possibility for DIET and other potential interactions between the two genera is required.
This study provides the first insight into the ecology of the A6 phylotype. Genomic evidence, coupled with their high relative abundance, suggests that members of the phylotype are important fermenters in mesophilic AD systems. The annotation of the representative CAMBI-1 genome gives a basic understanding of their physiology, noting that most of the coding sequence was not associated with any function ( Table 1). A detailed understanding of the ecology of the A6 will be achieved with in situ and gene expression studies and by obtaining axenic cultures for representatives of the genus. The attainment of a genome and the optimisation of FISH probes in this study provides an important foundation for these approaches. Importantly, having complete genomes representing the abundant members of the community is an essential reference for metatranscriptomic and metaproteomic gene expression studies that will together allow organism-based metabolic networks to be developed for anaerobic digester systems -giving a comprehensive view of the ecology of these biotechnologically important systems (Waldor et al., 2015).

TAXONOMIC PROPOSAL
In the absence of a pure culture, and with only partial characterisation, organisms have previously been given provisional Candidatus status names (Murray and Stackebrandt, 1995). However, in accordance with the recent recommendations of Whitman (2016), regarding the naming of uncultured organisms where the genome sequence is known, we propose the name Brevefilum fermentans gen. nov. sp. nov. with the CAMBI-1 genome as a provisional type species reference.

AUTHOR CONTRIBUTIONS
SM, RK, and PN planned the experimental work. SM and EF performed the FISH-based analyses. RK performed the DNAbased experimental work. RK, SK, and MA performed the metagenomic analyses. SM and MD performed the genome annotation. The manuscript was written by SM and drafted by all authors.

FUNDING
This work was supported by the Danish Council for Independent Research (grant no. 4093-00127A), the Villum Foundation and Aalborg University.