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 micro-organisms, resulting in the decomposition of complex organic substances into simple compounds (mainly CH4 and CO2) [2]. Since the beginning of the use of culture-independent techniques, increasing numbers of ecological studies have indicated that the phylum is one of the predominant and widespread bacterial groups in various anaerobic digesters [3-6]. It is well known that groups of the order in the phylum (such as , , and ) are some of the most common hydrolytic bacteria in anaerobic bioreactors, especially in cellulolytic environments [7-11]. , belonging to the order of the phylum , was erected by Jabari [12] to describe thermophilic, anaerobic, Gram-positive, rod-shaped, non-motile, terminal-spore-forming and saccharolytic bacteria. LIND6LT2T was isolated from an upflow anaerobic digester treating waste water, and was assigned as the type species of the family .

We collected samples from an anaerobic batch digester treating animal manure and rice straw, which was pre-enriched 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 , and uncultured were the predominant organisms of the microbial consortium (unpublished data). To reveal the ecophysiological roles of anaerobic bacteria in anaerobic digestion, strain A6T 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): NH4Cl, 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 % N2. 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) rice-straw-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 [14-16]. 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 A6T. 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 A6T, d-glucose was used as the main substrate, instead of acetate or propionate. The taxonomic description of strain A6T 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 neighbour-joining 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 A6T belonged to the family , and that its closest relative was LIND6LT2T (96 % sequence similarity), followed by AP3T (88.9 %), Ra1766G1T (88.4 %), ED-Mt61/PYG-s6T (88.3 %) and LMG 24724T (88.11 %) (Fig. 1).

Fig. 1.

Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences showing the relationship between strain A6T and its phylogenetically close relatives. The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain A6T is KF766957 (1413 bp). Bar, 0.02 changes per nucleotide position.

The cultural and morphological characteristics of the isolated strain A6T 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−1) and trypticase (0.5 g l−1). Prior to inoculation, filter-sterilized glucose solution was added as substrate (final concentration 5 g l−1) 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 phase-contrast microscope (Nikon 80i).

DSM 22681T was obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) for comparson of its physiological and chemotaxonomic characteristics with those of strain A6T. Growth experiments to determine the pH, temperature and NaCl concentration ranges were performed in triplicate using Hungate tubes with 5 ml of BCTY medium containing glucose as the substrate. The pH range examined for growth was 5.5–10.0, and was adjusted using the following sterile anaerobic solutions (20 mM): MES (5.5, 6.0), PIPES (6.5, 7.0, 7.5), HEPES (8.0), Tricine (8.5) and CHES (9.0, 9.5, 10.0). The temperature range investigated was 35–65 °C at 5 °C intervals, and the NaCl concentration range was 0–25.0 g NaCl l−1. Substrate utilization tests were performed in basal medium with d-glucose, d-xylose, maltose, d-fructose, d-galactose, d-ribose, d-sucrose, d-lactose, d-mannose, d-mannitol, raffinose, l-rhamnose, cellobiose, d-arabinose, yeast extract, acetate, propionate, pyruvate and lactate. Each substrate was added at a final concentration of 20 mM (for sugars and organic acids). The strain was subcultured at least twice under the same experimental conditions prior to determination of growth rates. Elemental sulfur (1 %, w/v), sulfate (20 mM), thiosulfate (20 mM), sulfite (2 mM), nitrate (10 mM) and nitrite (2 mM) were tested as terminal electron acceptors. 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 m×320 µm×0.25 µm) and a flame ionization detector with N2 as the carrier gas at a flow rate of 36 ml min−1. H2 and CO2 were analysed by GC (Agilent 7820A) using a porapak Q packed column (2 m×30 µm) and thermal conductivity detector with N2 as the carrier gas at a flow rate of 30.0 ml min−1 and column temperature of 80 °C. H2S 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 Na2CO3 and 0.3 mM NaHCO3 as eluent at a flow rate of 1.2 ml min−1.

Strain A6T formed white and round colonies after 2 days at 55 °C. Cells were non-motile and slightly curved rods with a size of 2.5–7.6×1–0.58 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−1 when glucose was used as the substrate in BCTY medium under the above-mentioned optimum conditions.

Fig. 2.

(a) Scanning electron micrograph of cells of strain A6T. (b) Transmission electron micrograph of thin sections of cells cultured for 24 h. Bars, 1 µm (a), 0.5 µm (b).

It is noteworthy that the addition of yeast extract enhanced growth of strain A6T. Similar to LIND6LT2T, growth of strain A6T 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, l-rhamnose, peptone, acetate, propionate, pyruvate or lactate. Moreover, cellulose was not hydrolysed by strain A6T. The fermentation products of the strain in a saccharide-utilizing culture were H2, CO2, 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 A6T were C16 : 0 (30.6 %), iso-C17 : 1 (30.3 %), C14 : 0 (18.1 %), C16 : 1ω7c (5.6 %), C16 : 0 N-alcohol (3.2 %), C13 : 0 3-OH (2.9 %), C13 : 1 AT 12–13 (1.0 %), C18 : 0 (0.8 %), C12 : 0 (0.7 %), C18 : 1ω7c (0.4 %), C16 : 1ω5c (0.3 %), C18 : 1ω9c (0.3 %) and an unknown component (5.7 %) (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 A6T was 36.6 mol%, which is similar to that of LIND6LT2T (35.2 mol%) [12].

Table 1.

Comparison of the cellular fatty acid profiles of strain A6T with its phylogenetically closest relative

Fatty acid	Strain A6T	D. saccharophila LIND6LT2T
C12 : 0	0.7	0.4
C13 : 1 AT 12–13	1.0	–
C14 : 0	18.1	8.3
C13 : 0 3-OH	2.9	–
C16 : 0	30.6	68.4
C16 : 0 N-alcohol	3.2	0.7
C16 : 1ω7c	5.6	–
C16 : 1ω5c	0.3	5.3
iso-C17 : 1	30.3	–
C18 : 1ω9c	0.3	0.8
C18 : 1ω7c	0.4	4.1
C18 : 0	0.8	7.3
Unknown	5.7	1.4

Although strain A6T was found to be phenotypically comparable to LIND6LT2T 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 LIND6LT2T, strain A6T 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 A6T could be easily distinguished from , , and by growth temperature and DNA G+C content. In addition, strain A6T and could also be differentiated based on the utilization of pectinous substrates.

Table 2.

Phenotypic comparison of strain A6T with its five phylogenetically closest relatives

Taxa: 1, strain A6T; 2, [12]; 3, [28]; 4, [29]; 5, [30]; 6, [31]. +, Positive; −, negative or very weakly positive; nd, not done. APL, aminophospholipid; DPG, diphosphatidylglycerol; GL, glycolipid; PG, phosphatidylglycerol; PGL, phosphoglycolipid; PL, phospholipid; L, unknown lipid; EtOH, ethanol; A, acetate; P, propionate; B, butyrate; iB, isobutyrate; F, formate; V, valerate; iV, isovalerate; L, lactate.

Characteristic	1	2	3	4	5	6
Gram stain	+	+	+	−	+	+
Morphology	Slightly curved rods (1–0.58×2.5–7.6 µm)	Rods (0.5×5–10 µm)	Rods with variable length (0.25–3×3–10 µm)	Rods (0.5–1×2–10 µm)	Rods (1.0–1.5×1.5–3.0 µm)	Long rods (5–15 µm)
Temperature (optimum) (°C)	50	50–55	43 (max.)	30–35	37	37–41
pH	7.5	7–7.5	9.5–9.7	6.5–7.5	5.5–9.3	6
NaCl concentration (%, w/v)	0	0.5	0.4–0.6 M Na+	2–3	nd
Motility	−	−	−	nd	−	nd
Major cellular fatty acids	C16 : 0, iso-C17 : 1, C14 : 0	C16 : 0, C14 : 0, C18 : 0	C16 : 0, C16 : 1ω7c, C18 : 1ω7c	anteiso-C15 : 0, iso-C15 : 0, anteiso DMA-C15 : 0, C16 : 0.	C16 : 0	nd
Polar lipids	PGL, PL, GL, DPG, PL, L	DPG, PG, PL, PGL, GL	PG, DPG, PL, GL, APL	DPG, PG, GL, PL	nd
DNA G+C content (mol%)	36.6	35.2	30.7	31.2	48	44
Substrates
d-Glucose	+	+	−	+	+	+
d-Xylose	+	+	−	+	−	−
d-Ribose	+	−	−	+	+	nd
d-Arabinose	+	−	−	+	−	−
d-Galactose	+	−	−	+	+	nd
d-Fructose	+	−	−	−	+	nd
Cellobiose	+	+	−	+	−	+
Sucrose	+	+	−	+	+	+
d-Lactose	+	−	−	−	−	−
d-Mannose	+	+	−	+	+	+
Maltose	+	+	−	+	−	+
Raffinose	+	−	−	+	+	+
d-Mannitol	−	+	−	−	−	+
l-Rhamnose	−	+	−	−	−	−
Others			Galacturonic acid, pectin, polygalacturonates	Pyruvate	Sorbitol, melibiose, melezitose	Salicin, sorbitol, trehalose
Fermentation end products	H2, CO2, EtOH, L, A, P, B, iB, V, iV,	H2, CO2, A, B, F, iB,	A, F	A	nd	B, A, P, H2, CO2

Therefore, based on the data from phylogenetic, physiological and chemotaxonomic analyses, strain A6T can be considered to represent a novel species of the genus belonging to the family , order , and phylum , for which we propose the name sp. nov.

Description of Defluviitalea raffinosedens sp. nov

(raf.fi.nos.e′dens. N.L. neut. n. raffinosum raffinose; L. pres. part. edens eating; N.L. part. adj. raffinosedens raffinose-eating).

Cells are Gram-stain-positive, slightly curved rods with a size of 2.5–7.6×1–0.58 µm, non-motile, occur singly or in pairs, and form spores at high temperature. Growth occurs at 40–65 °C (optimum 50 °C), pH 6.5–8.0 (optimum 7.5) and an NaCl concentration of 0–20 % (w/v). Cells are thermophilic and anaerobic, and hydrolyse d-glucose, maltose, d-xylose, d-galactose, d-fructose, d-ribose, lactose, d-mannose, raffinose, sucrose, d-arabinose, cellobiose and yeast extract. Yeast extract is required for growth. The major cellular fatty acids are C16 : 0, iso-C17 : 1, C14 : 0, C16 : 1ω7c, C16 : 0 N-alcohol and C13 : 0 3-OH. Respiratory quinones are not found. The polar lipids are phosphoglycolipids, phospholipids, glycolipids, a diphosphatidylglycerol and a phosphatidylglycerol.

The type strain is A6T (=DSM 28090T=ACCC 19951T), 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%.