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1 Department of Environmental Systems Engineering, Nagaoka University of Technology, Nagaoka, Niigata 940-2188, Japan
2 Subground Animalcule Retrieval (SUGAR) Program, Extremobiosphere Research Center, Japan Agency for Marine-Earth Science & Technology (JAMSTEC), Yokosuka, Kanagawa 237-0061, Japan
3 Department of Civil Engineering, Tohoku University, Sendai 980-8579, Japan
4 Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki 305-8566, Japan
5 Research Institute of Genome-Based Biofactory, National Institute of Advanced Industrial Science and Technology (AIST), Sapporo, Hokkaido 062-8517, Japan
Correspondence
Hiroyuki Imachi
imachi{at}jamstec.go.jp
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene and dsrAB gene sequences of strain MGPT are AB154390 and AB154391, respectively.
A figure showing the effect of temperature, pH and NaCl concentration on the specific growth rate of strain MGPT is available with the online version of this paper.
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; Kaspar & Wuhrmann, 1978; Mah et al., 1990). Under methanogenic conditions, propionate degradation is carried out by a syntrophic association of propionate-oxidizing, hydrogen (and/or formate)-producing fermentative bacteria and hydrogenotrophic methanogens, because the oxidation of propionate is thermodynamically unfavourable in such environments unless the consumption of the reducing equivalents (hydrogen and/or formate) is coupled with the oxidation (Schink, 1997). Due to this thermodynamic limitation, propionate-degrading anaerobes can gain only a small amount of energy through the syntrophic reaction, and thus these organisms grow very slowly. Because of these traits, isolation of propionate-degrading bacteria has been difficult. To date, eight species have been isolated and described. Five species belong to the class Deltaproteobacteria; these include Syntrophobacter wolinii (Boone & Bryant, 1980), Syntrophobacter pfennigii (Wallrabenstein et al., 1995), Syntrophobacter fumaroxidans (Harmsen et al., 1998), Syntrophobacter sulfatireducens (Chen et al., 2005) and Smithella propionica (Liu et al., 1999). The remaining three species belong to a Gram-positive bacterial lineage ‘Desulfotomaculum cluster I’ in the phylum Firmicutes: i.e. Desulfotomaculum thermobenzoicum subsp. thermosyntrophicum (Plugge et al., 2002), Pelotomaculum thermopropionicum (Imachi et al., 2002) and Pelotomaculum schinkii (de Bok et al., 2005). All the deltaproteobacterial species and P. schinkii are mesophiles; the others are thermophiles.
Recently, we reported the isolation of a novel anaerobic, mesophilic, propionate-oxidizing bacterium, designated strain MGPT, as a defined co-culture with a hydrogenotrophic methanogen, Methanospirillum hungatei (Imachi et al., 2006). 16S rRNA gene sequence analysis revealed that the strain was affiliated with ‘Desulfotomaculum subcluster Ih’, which consists of the genera Pelotomaculum and Cryptanaerobacter. In addition, strain MGPT was found to have dsrAB genes encoding the 
and 
subunits of dissimilatory (bi)sulfite reductase, although the strain could not reduce sulfate, sulfite or organosulfonates, which are typical electron acceptors for dissimilatory sulfate-reduction. In this report, we describe detailed morphological and physiological characteristics of strain MGPT and propose that this strain is a novel species of the genus Pelotomaculum.
Strain MGPT was originally isolated in co-culture with Methanospirillum hungatei from the methanogenic sludge of a mesophilic upflow anaerobic sludge blanket (UASB) reactor used to treat artificial wastewater, which was mainly composed of sucrose, propionate and acetate, as described by Sekiguchi et al. (1998, 1999). Methanospirillum hungatei strain JF1 (DSM 864) was purchased from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ, Braunschweig, Germany). We also used a hydrogen- and formate-utilizing methanogen, Methanothermobacter thermautotrophicus strain type-II, isolated in our laboratory. The media for cultivation of strain MGPT were prepared as described previously (Sekiguchi et al., 2000). Methanospirillum hungatei and Methanothermobacter thermautotrophicus were cultivated using the same medium as for strain MGPT, except that hydrogen (approx. 100–150 kPa) and acetate (1 mM) were added to the vials as the energy and carbon sources, respectively. All incubations were performed at 37 °C in 50 ml serum vials containing 20 ml medium (pH at 25 °C, 7.2) under an atmosphere of N2/CO2 (80 : 20, v/v) without shaking, unless otherwise noted. Neutralized substrates were added to the vials from stock solutions prior to inoculation. Growth and substrate utilization were determined by monitoring turbidity visually and by the production of methane, hydrogen and acetate. All incubations for the substrate tests were performed using growing cultures (10 %, v/v, inoculum) at 37 °C for over 3 months. The effects of pH, temperature and NaCl concentration on the growth of strain MGPT in co-culture with Methanospirillum hungatei were determined by using 20 mM propionate plus 0.01 % yeast extract medium with co-cultured cells of strain MGPT in exponential phase used as the inoculum. For determination of the optimum pH for growth, the pH value of the propionate medium was adjusted at room temperature to 5.5–8.0 with HCl or NaOH solutions under 100 % N2 gas prior to inoculation. To determine the temperature range for growth, cultures were incubated at 20–60 °C (pH 7.2). To evaluate the effect of NaCl concentration on growth, an autoclaved NaCl solution was added to the medium to give final concentrations of 1–20 g NaCl l–1. All measurements were performed in triplicate, and the specific growth rate was calculated based on methane production. Cell morphology was examined under a fluorescent microscope (Olympus BX50F). The Gram-staining reaction was performed by Hucker's method (Doetsch, 1981). Transmission electron microscopy was performed with a Hitachi H7000 transmission electron microscope as described by Hattori et al. (2000). Short-chain fatty acids, sulfate, sulfite, alcohols, methane, hydrogen and carbon dioxide were measured as described previously (Imachi et al., 2000, 2002, 2006). Comparative 16S rRNA sequence phylogenetic analysis was performed as described previously (Imachi et al., 2006). Bootstrap resampling analysis was performed with neighbour-joining, maximum-parsimony and maximum-likelihood methods to estimate the confidence of tree topologies as described previously (Sekiguchi et al., 2006).
Colony isolation was first attempted by the roll tube method on propionate medium in the presence of Methanospirillum hungatei, but the attempt was unsuccessful. Therefore, the culture was purified by repetitive serial dilutions and heat treatments. The purity of the strain MGPT culture was checked on several different test media (Imachi et al., 2006). Moreover, we also tested the purity by 16S rRNA gene-based cloning analyses using the universal bacterial primer pair EUB338*/1490R (Hatamoto et al., 2007; Weisburg et al., 1991) and the universal archaeal primer pair Ar109f/1490R (Imachi et al., 2002; Weisburg et al., 1991). Thirty rRNA gene clones were randomly selected and sequenced from respective clone libraries. The result of the molecular survey also demonstrated that the MGPT culture consisted of strain MGPT and Methanospirillum hungatei. The co-cultured cells converted propionate to methane and acetate nearly stoichimetrically (1 : 0.75 : 1; molar ratio), indicating that strain MGPT may use the methylmalonyl-CoA pathway, which is known to be one of the propionate-oxidizing pathways (Kosaka et al., 2006). To investigate whether other substrates support growth of and are utilized by strain MGPT, various substrates were tried with the co-cultured cells as the inoculum. The following substrates did not support growth and were not utilized (substrates were added at a final concentration of 20 mM unless otherwise indicated): Casamino acids (0.1 %), yeast extract (0.4 %), tryptone (0.1 %), crotonate, H2/CO2 (80 : 20, v/v, head space) plus acetate (1 mM) plus 2-bromoethanesulfonic acid (2-BES) (5 mM), betaine (10 mM), glucose, ribose, xylose, lactate, glycerol (5 mM), formate (40 mM) plus acetate (1 mM) plus 2-BES (5 mM), acetate, butyrate, valerate (5 mM), caproate (5 mM), heptanoate (5 mM), malate, fumarate, succinate, ethanol (10 mM), methanol, 1-propanol, 1-butanol, 1-pentanol (10 mM), acetoin (10 mM), acetaldehyde (0.1 %), 1,2-butanediol (10 mM), 2,3-butanediol (10 mM), 1,3-propanediol (10 mM), ethylene glycol (10 mM), arabinose, 2-propanol, fructose, galactose, mannose, raffinose, sucrose, starch (5 g l–1), xylan (5 g l–1), pectin (5 g l–1), 2-oxoglutarate (10 mM), proline (10 mM), glycine (10 mM), aspartate (10 mM), serine (10 mM), alanine (10 mM), glutamate (10 mM), 3-hydroxybutyrate (10 mM), 4-hydroxybutyrate (10 mM), 1,3-benzenediol (1 mM), benzoate (5 mM), 2,3-dihydroxybenzoate (1 mM), 2,4-dihydroxybenzoate (1 mM), 2,5-dihydroxybenzoate (1 mM), hydroquinone (1 mM), m-hydroxybenzoate (1 mM), p-hydroxybenzoate (1 mM), vanillate (1 mM), syringate (1 mM), acrylate (1 mM), o-methoxybenzoate (1 mM), phthalate and its isomers (1 mM), p-toluic acid (1 mM), 3-chlorobenzoate (1 mM) and phenol (1 mM). In addition, the following electron acceptors were not utilized for growth in the presence of propionate and 2-BES (5 mM): sulfate, sulfite (2 mM), thiosulfate, elemental sulfur, taurine (10 mM), isethionate (10 mM), cysteate (5 mM), nitrate, ferric ion [Fe(III)-nitrilotriacetate (NTA): 5 mM], fumarate, anthraquinone disulfonic acid (10 mM), crotonate, proline, glycine, 4-hydroxybenzoate (10 mM) or oxygen. Therefore, we used co-cultured cells of strain MGPT for further physiological and genetic characterizations.
Cells of strain MGPT are sausage-shaped rods, non-motile, 2.0–4.0 µm long and 1.0 µm wide, and occur singly or in pairs (Fig. 1). The cells often form aggregates with cells of Methanospirillum hungatei. A similar finding was also reported for a syntrophic, propionate-degrading culture of Pelotomaculum thermopropionicum (Ishii et al., 2005). The cells are Gram-positive. Spores are spherical and central. Spore formation was observed during the late-exponential growth phase.
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). The pH range for growth and substrate utilization of strain MGPT was estimated to be 6.5–7.5 with an optimum of 6.5–7.2 (supplementary Fig. S1b). Under optimum conditions (pH 7.0, 37 °C), the specific growth rate on 20 mM propionate medium was approx. 0.2 per day (calculated by measuring methane production). The strain could grow at NaCl concentrations ranging from 0 to 5 g l–1 (supplementary Fig. S1c), while Methanospirillum hungatei could grow with 0–15 g NaCl l–1.
For strain MGPT, 1571 bp of the 16S rRNA gene sequence was determined. On the basis of the phylogenetic analysis described previously (Imachi et al., 2006), strain MGPT was affiliated with subcluster Ih of ‘Desulfotomaculum cluster I’. We reconstructed a phylogenetic tree that included 16S rRNA gene sequences from each subcluster containing cultured representatives (Fig. 2). The closest relatives of the 16S rRNA gene of strain MGPT were the rrnB 16S rRNA gene sequence of Pelotomaculum schinkii (de Bok et al., 2005) and that of Cryptanaerobacter phenolicus (Juteau et al., 2004) (both with similarity values of 95 %).
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), particularly the following two traits: (1) all members of the subcluster lack the ability to reduce sulfate, as does strain MGPT, although they belong to the sulfate-reducing Desulfotomaculum cluster; and (2) like almost all Pelotomaculum spp., strain MGPT grows syntrophically with hydrogenotrophic methanogen (C. phenolicus was not tested for syntrophic growth; Juteau et al., 2004). The most striking feature of strain MGPT is its obligately syntrophic life style with hydrogenotrophic methanogens. Obligate syntrophy is also found in some members of the genus Pelotomaculum. P. schinkii can grow only on propionate in co-culture with hydrogenotrophic methanogens and was thus reported as the ‘first true obligately syntrophic propionate-oxidizing bacterium’ (de Bok et al., 2005). Also, Pelotomaculum isophthalicicum grows only with aromatic compounds in co-culture with hydrogenotrophic methanogens (Qiu et al., 2006). Strain MGPT shares these phenotypes. However, 16S rRNA gene sequence similarity values support the novelty of strain MGPT at least at species level (Stackebrandt & Goebel, 1994). In addition to this genetic difference, one notable feature that distinguishes strain MGPT from the known members of the genus Pelotomaculum may be that strain MGPT possesses dsrAB genes (Table 1). These genotypic differences support the conclusion that strain MGPT is a novel species in the genus Pelotomaculum for which we propose the name Pelotomaculum propionicicum sp. nov.
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Strictly anaerobic. The organism only grows syntrophically on propionate with hydrogenotrophic methanogens. Although the organism does not demonstrate sulfate reduction, it has dsrAB genes that are essential for sulfate respiration. Cells are 2.0–4.0 µm long and 1.0 µm wide, occurring singly or in pairs. Spores are spherical and central. Gram-positive. Temperature range for growth is 25–45 °C (optimum: 37 °C). The pH range for growth is 6.5–7.5 (optimum: 6.5–7.2). Growth occurs in the presence of 0–0.5 % NaCl, but does not occur in the presence of greater than 1.0 % NaCl. Habitat is an anaerobic, mesophilic granular sludge.
The type strain is MGPT (=DSM 15578T=JCM 11929T). The strain is stored in the DSM and JCM culture collections as a defined co-culture with Methanospirillum hungatei.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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