Defluviitalea raffinosedens sp. nov., a thermophilic, anaerobic, saccharolytic bacterium isolated from an anaerobic batch digester treating animal manure and rice straw

A thermophilic, anaerobic, fermentative bacterium, strain A6T, was obtained from an anaerobic batch digester treating animal manure and rice straw. Cells were Gram-stain-positive, slightly curved rods with a size of 0.6–1×2.5–8.2 µm, non-motile and produced terminal spores. The temperature, pH and NaCl concentration ranges for growth were 40–60 °C, 6.5–8.0 and 0–15.0 g l−1, with optimum growth noted at 50–55 °C, pH 7.5 and in the absence of NaCl, respectively. Yeast extract was required for growth. d-Glucose, maltose, d-xylose, d-galactose, d-fructose, d-ribose, lactose, raffinose, sucrose, d-arabinose, cellobiose, d-mannose and yeast extract were used as carbon and energy sources. The fermentation products from glucose were ethanol, lactate, acetate, propionate, butyrate, valerate, iso-butyrate, iso-valerate, H2 and CO2. The G+C content of the genomic DNA was 36.6 mol%. The predominant fatty acids were C16 : 0, iso-C17 : 1, C14 : 0, C16 : 1ω7c, C16 : 0 N-alcohol and C13 : 0 3-OH. Respiratory quinones were not detected. The polar lipid profile comprised phosphoglycolipids, phospholipids, glycolipids, a diphosphatidylglycerol, a phosphatidylglycerol and an unidentified lipid. Phylogenetic analyses of the 16S rRNA gene sequence indicated that the strain was closely related to Defluviitalea saccharophila DSM 22681T with a similarity of 96.0 %. Based on the morphological, physiological and taxonomic characterization, strain A6T is considered to represent a novel species of the genus Defluviitalea, for which the name Defluviitalea raffinosedens sp. nov. is proposed. The type strain is A6T (=DSM 28090T=ACCC 19951T).

Anaerobic digestion is a method of waste treatment aimed at reducing the hazardous effects of wastes on the biosphere [1]. It comprises complex, redox biochemical reactions driven by various anaerobic and relatively anaerobic microorganisms, resulting in the decomposition of complex organic substances into simple compounds (mainly CH 4 and CO 2 ) [2]. Since the beginning of the use of cultureindependent techniques, increasing numbers of ecological studies have indicated that the phylum Firmicutes is one of the predominant and widespread bacterial groups in various anaerobic digesters [3][4][5][6]. It is well known that groups of the order Clostridiales in the phylum Firmicutes (such as Clostridium, Acetivibrio, Selenomonas and Ruminococcus) are some of the most common hydrolytic bacteria in anaerobic bioreactors, especially in cellulolytic environments [7][8][9][10][11]. Defluviitaleaceae, belonging to the order Clostridiales of the phylum Firmicutes, was erected by Jabari [12] to describe thermophilic, anaerobic, Gram-positive, rodshaped, non-motile, terminal-spore-forming and saccharolytic bacteria. Defluviitalea saccharophila LIND6LT2 T was isolated from an upflow anaerobic digester treating waste water, and was assigned as the type species of the family Defluviitaleaceae.
We collected samples from an anaerobic batch digester treating animal manure and rice straw, which was preenriched with PY medium (2 g peptone and 1 g yeast extract per litre distilled water) containing rice straw (5 g per litre distilled water), and a microbial consortium degrading rice straw under anaerobic methanogenic conditions at 40 C was obtained and subcultured for 10 years. The 16S rRNA clone libraries and high-throughput sequencing analyses revealed that Clostridium, Gracilibacter, Sedimentibacter and uncultured Firmicutes were the predominant organisms of the microbial consortium (unpublished data). To reveal the ecophysiological roles of anaerobic bacteria in anaerobic digestion, strain A6 T was enriched and isolated from the above-mentioned microbial consortium at 55 C using enriched medium (basal medium containing 1 g yeast extract and 5 g sodium acetate or 3 g sodium propionate). The basal medium contained the following (per litre distilled water): NH 4 Cl, 1.0 g; yeast extract, 0.1 g; L-Cys-HCl, 1 g; 0.1 % (w/v) resazurin solution, 1.0 ml; macro mineral solution, 50.0 ml; trace mineral solution, 10.0 ml; and vitamin mix solution, 10.0 ml. The macro mineral solution, trace mineral solution and vitamin mix solution were prepared as described previously [13]. The agar medium was supplemented with 18.0 g agar. All the media were prepared and dispensed anaerobically under a gaseous atmosphere of 100 % N 2 . The pH of the medium was adjusted to 6.5-7.0 with 5 M KOH, and the media were sterilized by autoclaving at 121 C for 30 min.
The enriched medium was inoculated with 2 % (v/v) ricestraw-degrading microbial consortium and incubated for 1 week at 55 C. For isolation, the enrichment culture was serially diluted tenfold in Hungate tubes containing molten agar medium, and the tubes were rolled following the procedures of the Hungate roll-tube technique [14][15][16]. Subsequently, single colonies were picked and transferred into liquid medium under anaerobic conditions. The roll-tube procedure was repeated several times until a pure culture was obtained. A single white and round colony was obtained and designated as strain A6 T . This strain did not utilize acetate or propionate, but grew at low cell concentration in enriched medium, indicating that the yeast extract in the medium served as carbon and energy source during enrichment and isolation. For subsequent incubation of strain A6 T , D-glucose was used as the main substrate, instead of acetate or propionate. The taxonomic description of strain A6 T is reported here based on phenotypic and phylogenetic studies.
The extraction and purification of DNA, PCR amplification and sequencing of the 16S rRNA were performed as described by Huang [17]. The sequence obtained was submitted to NCBI for initial alignment with highly similar sequences in the BLASTN program. The 16S rRNA sequences from closely related organisms were retrieved from NCBI and EzTaxon. Phylogenetic trees were reconstructed with the software package MEGA version 5.0 using the neighbourjoining and maximum-likelihood methods [18]. The robustness of the topology in the phylogenetic tree was evaluated by bootstrap analysis based on 1000 replicates. Phylogenetic analysis based on the 16S rRNA gene sequence revealed that strain A6 T belonged to the family Defluviitaleaceae, and that its closest relative was D. saccharophila LIND6LT2 T (96 % sequence similarity), followed by Natranaerovirga pectinivora AP3 T (88.9 %), Vallitalea guaymasensis Ra1766G1 T (88.4 %), Lactonifactor longoviformis ED-Mt61/PYG-s6 T (88.3 %) and Anaerostipes butyraticus LMG 24724 T (88.11 %) (Fig. 1).
The cultural and morphological characteristics of the isolated strain A6 T were investigated using cells cultivated on basal carbonate yeast extract and trypticase medium (BCTY medium). The BCTY medium consisted of basal medium, yeast extract (0.5 g l À1 ) and trypticase (0.5 g l À1 ). Prior to inoculation, filter-sterilized glucose solution was added as substrate (final concentration 5 g l À1 ) to the sterile BCTY medium. Cell morphology was examined using a scanning electron microscope (JEOL JSM-7500F) and transmission electron microscope (Hitachi H-600IV). Gram staining was performed using the traditional method [19] and spore staining was performed conventionally [20]. The presence of spores and Gram staining were observed using a phasecontrast microscope (Nikon 80i). Growth was determined by measuring the turbidity of the cultures at a wavelength of 600 nm using a spectrophotometer (DU 730; Beckmann) as described previously [12].
The liquid fermentation products were determined by GC (Agilent 7890A) using an FFAP column (30 mÂ320 µmÂ0.25 µm) and a flame ionization detector with N 2 as the carrier gas at a flow rate of 36 ml min À1 . H 2 and CO 2 were analysed by GC (Agilent 7820A) using a porapak Q packed column (2 mÂ30 µm) and thermal conductivity detector with N 2 as the carrier gas at a flow rate of 30.0 ml min À1 and column temperature of 80 C. H 2 S production was determined photometrically as described by Cord-Ruwisch [21]. Sulfate, nitrate and nitrite were measured by ion chromatography (Dionex ICS-3000) using an IonPac AG12A column with 2.7 mM Na 2 CO 3 and 0.3 mM NaHCO 3 as eluent at a flow rate of 1.2 ml min À1 .
Strain A6 T formed white and round colonies after 2 days at 55 C. Cells were non-motile and slightly curved rods with a size of 2.5-7.6Â1-0.58 µm, occurring singly or in pairs (Fig. 2). Furthermore, the strain was Gram-stain-positive and formed spores at high temperature. The temperature, pH and NaCl concentration ranges for growth of the strain were 40-65 C (optimum 50 C), 6.5-8.0 (optimum 7.5) and 0-20 % (w/v) (optimum 0 %), respectively (Fig. S1, available in the online Supplementary Material). The maximum growth rate of the strain was 0.58 h À1 when glucose was used as the substrate in BCTY medium under the abovementioned optimum conditions.
It is noteworthy that the addition of yeast extract enhanced growth of strain A6 T . Similar to D. saccharophila LIND6LT2 T , growth of strain A6 T was improved with increasing concentrations of yeast extract. Elemental sulfur, thiosulfate, sulfite, sulfate and nitrate were not used as electron acceptors. While the strain was able to ferment D-glucose, maltose, D-xylose, D-galactose, D-fructose, D-ribose, lactose, raffinose, sucrose, D-arabinose, cellobiose, D-mannose and yeast extract, it could not utilize D-mannitol, Lrhamnose, peptone, acetate, propionate, pyruvate or lactate. Moreover, cellulose was not hydrolysed by strain A6 T . The fermentation products of the strain in a saccharide-utilizing culture were H 2 , CO 2 , ethanol, lactate, acetate, propionate, butyrate, valerate, traces of iso-butyrate, and iso-valerate.
The DNA G+C content, cellular fatty acid composition, respiratory quinones and polar lipids were evaluated by the Identification Service of the DSMZ (Braunschweig, Germany). The DNA G+C content was determined by using HPLC as described by Mesbah et al. [22]. The cellular fatty acid composition was determined by saponification, methylation and extraction as described earlier with minor modifications [23,24]. Fatty acids were analysed using the Sherlock MIS system (MIDI). Respiratory quinones were extracted using methanol/  hexane [25,26], followed by phase separation into hexane. Respiratory lipoquinones were separated by TLC on silica gel (Macherey-Nagel Art. No. 805023), using hexane/tetrabutylmethylether (9 : 1, v/v) as the solvent and further analysed by HPLC. Polar lipids were extracted using chloroform/methanol/0.3 % aqueous NaCl mixture (1 : 2 : 0.8, by vol.) and separated by two-dimensional silica gel TLC (Macherey-Nagel Art. No. 18135). The total lipid content was detected using the method described by Tindall et al. [27].
The major whole-cell fatty acids of strain A6 T were C 16 (Table 1). Respiratory quinones were not detected. The polar lipid profile comprised phosphoglycolipids, phospholipids, glycolipids, a diphosphatidylglycerol and a phosphatidylglycerol (Fig. S2). The DNA G+C content of strain A6 T was 36.6 mol%, which is similar to that of D. saccharophila LIND6LT2 T (35.2 mol%) [12].
Although strain A6 T was found to be phenotypically comparable to D. saccharophila LIND6LT2 T with respect to cell morphology, optimum pH and temperature for growth, electron acceptors, and polar lipid profile, it differed with respect to major cellular fatty acids and substrate utilization. Unlike D. saccharophila LIND6LT2 T , strain A6 T did not ferment D-mannitol or L-rhamnose, but fermented D-galactose, D-fructose, D-ribose, lactose, raffinose and D-arabinose    (Table 2). Moreover, strain A6 T could be easily distinguished from N. pectinivora, V. guaymasensis, L. longoviformis and A. butyraticus by growth temperature and DNA G+C content. In addition, strain A6 T and N. pectinivora could also be differentiated based on the utilization of pectinous substrates.
Therefore, based on the data from phylogenetic, physiological and chemotaxonomic analyses, strain A6 T can be considered to represent a novel species of the genus Defluviitalea, belonging to the family Defluviitaleaceae, order Clostridiales, and phylum Firmicutes, for which we propose the name Defluviitalea raffinosedens sp. nov.
The type strain is A6 T (=DSM 28090 T =ACCC 19951 T ), isolated from an anaerobic batch digester treating animal manure and rice straw. The G+C content of the genomic DNA of the type strain is 36.6 mol%.

Funding information
This work was supported by the Agricultural Science and Technology Innovation Program (ASTIP), Chinese Academy of Agricultural Sciences, and the Science Infrastructure Platform of Sichuan Province Science and Technology Support Program (Grant TJPT20160013).