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1 Institute of Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology (AIST), Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan
2 Department of Environmental Systems Engineering, Nagaoka University of Technology, Nagaoka, Niigata 940-2188, Japan
3 Division of Industrial and Environmental Physics, Indonesian Institute of Sciences, Kompleks LIPI, Bandung 40135, Indonesia
Correspondence
Yuji Sekiguchi
y.sekiguchi{at}aist.go.jp
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Photomicrographs of strains JE and OL cultivated anaerobically with sucrose and yeast extract at 45 °C are available as supplementary material in IJSEM Online.
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TOPABSTRACTMAIN TEXTREFERENCES |
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; Stams, 1994). Syntrophic association between two metabolic groups, such as syntrophic fatty-acid-oxidizing bacteria and hydrogenotrophic methanogens, is a typical example of such symbioses that can be found ubiquitously in methanogenic ecosystems. The isolation of ‘S organism’ from ‘Methanobacillus omelianskii’ cultures in 1967 provided the first tangible evidence of syntrophic substrate degradation (Bryant et al., 1967). Although the ‘S organism’ was lost and hence its exact taxonomic placement remains unknown, the organism was characterized as a Gram-negative, strictly anaerobic, oval-rod-shaped bacterium that could catalyse syntrophic degradation of ethanol and other primary aliphatic alcohols (Reddy et al., 1972). Since the discovery of ‘S organism’, a number of species catalysing the syntrophic degradation of primary alcohols has been cultivated and characterized (Schink, 1997). In addition to ‘S organism’ and other unnamed strains (Plugge et al., 1990; Wu et al., 1992), sulphate-reducing species of the genus Desulfovibrio (Bryant et al., 1977) and some members of the genera Pelobacter (Schink & Stieb, 1983; Schink, 1984, 1985), Acetobacterium (Eichler & Schink, 1984) and Syntrophobacter (Wallrabenstein et al., 1995) strains perform syntrophic alcohol degradation in co-culture with hydrogenotrophic methanogens under mesophilic conditions. Furthermore, some members of the genera Desulfotomaculum (Klemps et al., 1985), Thermoanaerobacter (Ben-Bassat et al., 1981) and Pelotomaculum (Imachi et al., 2002) degrade primary alcohols syntrophically under thermophilic conditions. It has been reported that ethanol degradation in methanogenic ecosystems, particularly in those under thermophilic conditions, is often an important step in methanogenesis, accounting for a large proportion of electrons derived from degradation of complex organic compounds (Stams & Zehnder, 1990). Since natural and man-made methanogenic ecosystems contain a vast array of uncultured bacteria (Sekiguchi & Kamagata, 2004), it is important to elucidate those species responsible for major degradation steps in the ecosystem and to expand our knowledge further of the diversity of syntrophic microbes residing in various anaerobic environments. Therefore, we attempted to cultivate various novel syntrophs from a variety of anaerobic ecosystems. During the surveys of novel syntrophic organisms, three novel strains capable of degrading primary aliphatic alcohols and lactate in co-culture with hydrogenotrophic methanogens were found. In the present study, detailed morphological, physiological and chemotaxonomic characteristics of the strains isolated are reported and a novel species and genus are proposed to accommodate them.
Strains JLT and JE were originally obtained from sludge of a full-scale thermophilic (55 °C) digester that decomposed municipal solid wastes (sludge J, from Niigata, Japan). Strain OL was isolated from sludge of a full-scale digester that degraded sewage sludge under thermophilic (55 °C) conditions (sludge O, from Osaka, Japan). Methanothermobacter thermautotrophicus strain 
HT (=DSM 1053T) was obtained from the DSMZ, Braunschweig, Germany. Methanothermobacter thermautotrophicus strain type II was isolated and maintained in our laboratory. The basal medium used for enrichment, isolation and maintenance of strains JLT, JE and OL was prepared based on that of Widdel & Pfennig (1981). The medium composition was as described previously (Sekiguchi et al., 2000). Primary enrichment cultures were incubated anaerobically at 55 °C. All incubations were carried out at 45 °C in 50 ml serum vials containing 20 ml medium (pH25 °C 7.0) under an atmosphere of N2/CO2 (80/20, v/v), unless otherwise mentioned. Neutralized substrates were added to vials containing basal medium from stock solutions prior to inoculation. Solid medium was prepared by adding purified agar (Agar noble; Difco) to the medium as described above at a final concentration of 20 g l–1. M. thermautotrophicus strains were cultivated at 55 °C using the same media used for strains JLT, JE and OL except that hydrogen (approx. 0.5–1.0 atm or 50–100 kPa) was added to the gas phase (80/20, v/v, N2/CO2) in the vials as an energy source.
To determine the optimum pH for growth of strains JLT, JE and OL, the pH of the autoclaved medium containing 20 mM sucrose and 0.01 % yeast extract was adjusted at room temperature to pH 5.0–9.0 by adding HCl or NaOH under a 100 % N2 atmosphere prior to inoculation. During incubation, the pH of the medium was routinely monitored to check whether the initial pH conditions had changed. To evaluate the optimum temperature for growth, isolates were cultivated anaerobically in sucrose plus yeast extract medium (pH25 °C 7.0) at 17, 20, 25, 37, 45, 50, 55, 60, 70 and 80 °C. Under all conditions, duplicate cultures (1 % inoculum) were used and OD400 was measured. To test growth and substrate utilization, autoclaved or filter-sterilized substrates were added to the medium. All cultures were incubated anaerobically at 55 °C, pH25 °C 7.0 for at least 4 weeks. All substrates, including Fe(III) NTA (nitrilotriacetate) (Roden & Lovley, 1993), were prepared as described previously (Sekiguchi et al., 2000). Growth and substrate utilization of the strains were determined by monitoring the increase in turbidity (OD400) and the production of acetate. In syntrophic growth/substrate utilization tests, M. thermautotrophicus cells were added to the medium (2 % inoculum); growth and substrate utilization were checked by measuring turbidity (OD400) and methane production.
Cell morphology was examined under a phase-contrast microscope (Olympus AX80T). The Gram-staining reaction was performed by Hucker's method (Doetsch, 1981). Phase-contrast micrographs were taken using wet mounts on agar-coated slides (Pfenning & Wagener, 1986) for exponential-phase cultures. Cells for thin-section electron microscopy were prepared as described previously (Sekiguchi et al., 2003). Short-chain fatty acids, methane, hydrogen and carbon dioxide were determined by GC (Sekiguchi et al., 2000). Determination of alcohols and other compounds was performed by HPLC as described previously (Imachi et al., 2002). For G+C content determination, DNA was extracted and purified according to Kamagata & Mikami (1991). The G+C content was determined by HPLC (Shimadzu LC-6A) with a UV detector (Shintani et al., 2000). A mean value was calculated from two independent experiments. Quinones were determined as described previously (Zhang et al., 2000). Cells for quinone analysis were harvested from cultures grown on medium containing lactate and thiosulphate. Fatty acids of cells were converted to methyl esters using HCl/methanol and identified by GC-MS (Hitachi M7200A FC/3DQMS system) (Hanada et al., 2002). Cells were harvested from cultures grown on the medium containing sucrose plus yeast extract at 45 °C.
For 16S rRNA gene sequencing, genomic DNA of the strains, as well as that from enrichment cultures, was recovered according to the method of Hiraishi (1992). 16S rRNA genes were amplified by PCR with Taq polymerase (Perkin Elmer) as described previously (Sekiguchi et al., 2000). The PCR primers used in the amplification were the bacterial domain universal primer 8F (5'-AGAGTTTGATCCTGGCTCAG-3'; positions 8–27 in Escherichia coli) and the prokaryote universal primer 1490R (5'-GGTTACCTTGTTACGACTT-3'; 1491–1509 in E. coli) (Weisburg et al., 1991). The PCR product was sequenced directly on a Beckman CEQ8000 DNA sequencer using a CEQ DTC quick start kit (Beckman Coulter). Sequence data were aligned in an ARB dataset using the ARB program package and aligned data were corrected manually using the editing tool in the package. Phylogenetic trees based on 16S rRNA gene sequences were constructed by the neighbour-joining method (Saitou & Nei, 1987) with the ARB program package (Ludwig et al., 2004). Bootstrap resampling analysis (Felsenstein, 1985) for 1000 replicates was performed with the neighbour-joining, maximum-parsimony and maximum-likelihood methods to estimate the confidence of tree topologies as described previously (Zhang et al., 2003) with slight modifications, i.e. the neighbour-joining and the maximum-parsimony methods were used with the PAUP* 4.0 package (Swofford, 2003) and the maximum-likelihood method was used with the TREEFINDER program package (Jobb et al., 2004).
Thermophilic sludges (55 °C) were taken from two different types (sludges J and O) of full-scale anaerobic digesters fed with organic solid wastes. Gently washed and homogenized sludges were used for primary enrichments using 20 mM lactate or 20 mM ethanol as substrate and the cultures were incubated anaerobically at 55 °C (enrichment JL, lactate culture with sludge J; enrichment JE, ethanol culture with sludge J; enrichment OL, lactate culture with sludge O; enrichment OE, ethanol culture with sludge O). Growth and methane production were observed from all four cultures within a week of inoculation. The cultures were further enriched by successive transfers using a 1 % inoculum. All the enrichments stably converted their respective substrates into acetate and methane over ten successive transfers. Enrichments JL, JE and OL contained at least two major cell morphotypes. One was a non-motile, F420-autofluorescent rod morphologically resembling species in the genus Methanothermobacter and the other was an irregular rod (identified later as a syntrophic bacterium). Enrichment OE contained cells with morphotypes that differed from those of cells of enrichments JL, JE, and OL. Further study was conducted only for enrichments JL, JE and OL. To purify the irregular rods in the three enrichment cultures, an attempt was made to cultivate them in pure culture with other substrates that could support their growth in axenic culture (without methanogens). Highly purified cultures of enrichments JL, JE and OL after more than ten successive transfers were serially diluted and inoculated in media containing various substrates, such as pyruvate, glucose, crotonate and lactate plus sulphate. In the presence of 0.01 % yeast extract, various carbohydrates such as glucose and sucrose supported growth of rod-shaped cells, the morphology of which was very similar to that of the irregular rods in the original enrichment cultures. Growth of cultures on the glucose/yeast extract medium could be observed within 2–3 days of incubation at 55 °C. After three successive transfers on the glucose/yeast extract medium, roll-tube isolation was performed. Small colonies that were white, lens-shaped and 0.5 mm in diameter formed after 1 week of incubation. This step was repeated several times and the purified strains, designated JLT, JE and OL, were obtained from enrichments JL, JE and OL, respectively.
Strain JLT cells were irregular rods, 1.5–10.0 µm in length and 0.6–0.8 µm wide (Fig. 1a). Cells of strain JE were 1.5–40.0 µm long and 0.6–0.8 µm wide (see Supplementary Fig. S1 in IJSEM Online) and cells of strain OL were 1.5–10.0 µm long and 0.6–0.8 µm wide (Supplementary Fig. S1). The length of the cells seemed to depend on the growth phase. Spore formation was not observed and Gram-staining was negative for all the isolates. Motility was not observed with any isolate. Electron microscopy demonstrated that cells of all three isolates possessed a Gram-positive-type cell wall (Fig. 1b).
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In co-culture with hydrogenotrophic methanogens M. thermautotrophicus strain HT or type II, the organism could grow at 45 °C and pH 7.0 with ethanol (20 mM), glycerol (5 mM) and lactate (20 mM). Since cells of strain HT could not utilize formate as an energy source (though strain type II could), interspecies formate transfer did not seem to be essential for the syntrophic growth of strain JLT. As shown in Fig. 2(a), 20 mM lactate was degraded almost completely and transformed into acetate and methane within 2 weeks of incubation in co-culture with hydrogenotrophic methanogens (electron recovery, 92 %; carbon recovery, 79 %, on the basis of lactate consumed and methane and acetate formed). However, in pure culture, acetate and hydrogen production from lactate stopped when hydrogen accumulated at approximately 0.02 atm (2 kPa) and lactate was not degraded further (Fig. 2b). Acetate, propionate, butyrate, methanol, 1-propanol, 1-butanol, 1,3-propanediol, 2,3-butanediol (all at 20 mM) and benzoate (5 mM) did not support growth of syntrophic cultures of strain JLT. The following compounds were tested as electron acceptors with lactate and 0.01 % yeast extract medium (45 °C, pH 7.0): 20 mM sulphate, 20 mM nitrate, 1 mM sulphite, 20 mM thiosulphate and 5 mM Fe(III) NTA. Only thiosulphate supported growth of strain JLT.
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. As shown in Table 1, there were few differences in phenotypic features of the three strains. A few differences in substrate utilization were found among the strains, e.g. utilization of crotonate, ribose, malate, arabinose, galactose and raffinose. All strains utilized the same substrates (primary alcohols and lactate) in co-culture with methanogens. All strains used thiosulphate as an electron acceptor. In addition, all strains had similar growth responses to pH and temperature.
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9) cis (11 %) and unsaturated derivatives of C15 : 1 (25 %) as the major fatty acids; minor fatty acids were C14 : 0 (6 %), C15 : 0 (3 %), C16 : 0 (6 %), unsaturated isomers of C17 : 1 (11 %), C17 : 0 (3 %), C18 : 0 (1 %), C13 : 0 (1 %), C18 : 1 (9) cis (2 %), an unsaturated isomer of C16 : 1 (3 %) and an unsaturated isomer of C14 : 1 (5 %). The major fatty acids of cells of strain JE were iso-C15 : 0 (39 %), C16 : 0 (14 %) and unsaturated derivatives of C15 : 1 (15 %); minor fatty acids were C14 : 0 (8 %), C15 : 0 (6 %), C17 : 0 (5 %), unsaturated isomers of C17 : 1 (4 %), C18 : 0 (3 %), C16 : 1 (9) cis (3 %), C13 : 0 (1 %), an unsaturated isomer of C16 : 1 (1 %) and an unsaturated isomer of C14 : 1 (1 %). The major fatty acids of cells of strain OL were iso-C15 : 0 (21 %), C17 : 0 (13 %), C16 : 0 (13 %) and unsaturated derivatives of C15 : 1 (12 %); minor fatty acids were C15 : 0 (9 %), C14 : 0 (8 %), unsaturated isomers of C16 : 1 (9) cis (6 %), C17 : 1 (6 %), C18 : 1 (9) cis (4 %), C18 : 0 (3 %), an unsaturated isomer of C16 : 1 (2 %), an unsaturated isomer of C14 : 1 (2 %) and C13 : 0 (1 %). Quinones were not detected in any of the strains.
For strains JLT, JE and OL, a total of 1451 nt of the 16S rRNA gene was sequenced and phylogenetic analyses revealed that the strains belong to a deeply branched lineage of the phylum Firmicutes, forming a coherent clade with members of the genera Thermovenabulum and Thermosediminibacter (Fig. 3). The most closely related species were Thermovenabulum ferriorganovorum (Zavarzina et al., 2002) (16S rRNA gene sequence similarity of 88 %) and species of the genus Thermosediminibacter (87 % similarity) (Fig. 3), indicating the novelty of our strains at the genus level. All the isolated strains were phylogenetically very closely related to each other (99–100 % similarity). Direct sequencing of bacterial 16S rRNA genes from the highly purified enrichment cultures (JL, JE and OL) indicated that the cultures contained bacteria whose 16S rRNA genes matched perfectly with those of the isolates in this study as the major bacterial population. This fact suggested that the strains obtained were the irregular rods found in the primary enrichment cultures.
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). Regarding the first type of metabolism, various thermophilic microbes capable of syntrophic degradation of lactate and primary alcohols have been described. For example, a thermophilic species of the sulphate-reducing genus Desulfotomaculum (Desulfotomaculum nigrificans) is also known to degrade ethanol and lactate in the absence of sulphate when the culture is co-cultivated with hydrogenotrophic methanogens (Klemps et al., 1985). Thermoanaerobacter brockii (formerly Thermoanaerobium brockii) was originally isolated as a thermophilic chemo-organotroph that could degrade various carbohydrates fermentatively (Cayol et al., 1995; Zeikus et al., 1979). Later, the organism was found to degrade ethanol only in co-culture with hydrogenotrophic methanogens (Ben-Bassat et al., 1981). However, syntrophic growth properties of other species within the genus Thermoanaerobacter have not been examined and, hence, the ability of other members of this genus to grow syntrophically is unknown. Our strains could utilize carbohydrates in addition to their ability to grow syntrophically. These traits seem to be physiologically similar to those of members of the genus Thermoanaerobacter rather than to those of other known syntrophic ethanol- and lactate-degraders. Members of the genus Thermoanaerobacter are also known to utilize thiosulphate as an electron acceptor, as observed in our strains (Jain & Zeikus, 1992). However, our strains were apparently distinct at the genus level from members of the genus Thermoanaerobacter in the following features: (i) the optimum growth temperature of our strains is much lower (45–50 °C) than those of members of the genus Thermoanaerobacter (65–75 °C) (Jain & Zeikus, 1992); (ii) our strains did not form a monophyletic group with members of the genus Thermoanaerobacter on the basis of 16S rRNA gene sequences; and (iii) the sequence similarities between 16S rRNA genes of our strains and Thermoanaerobacter species were less than 85 % (Fig. 3
). Phylogenetically, the most closely related species was Thermovenabulum ferriorganovorum, which was isolated from a terrestrial hydrothermal source as a thermophilic anaerobe (Zavarzina et al., 2002). Thermovenabulum ferriorganovorum can utilize various carbohydrates fermentatively. Although Thermovenabulum ferriorganovorum and our strains share some basic physiological traits (such as anaerobic and saccharolytic growth), the following notable differences between them were found (Table 1). Thermovenabulum ferriorganovorum cells are straight or sometimes branched rods that form spores, whereas our strains are straight rods or filaments that do not form spores. Thermovenabulum ferriorganovorum grows at 45–76 °C, with optimum growth at 63–65 °C, whereas our strains are moderately thermophilic (25–60 °C with an optimum of 45–50 °C). In addition, Thermovenabulum ferriorganovorum reduces various oxidized forms of sulphur, nitrogen and metals, whereas our strains can reduce thiosulphate only. Other related thermophilic chemo-organotrophs in the genus Thermosediminibacter also show physiological traits that are similar to those of our strains, such as fermentative growth with various monomeric carbohydrates (Lee et al., 2005). However, although similar to Thermovenabulum ferriorganovorum, there are a number of phenotypic differences between our strains and these anaerobes (as shown in Table 1). On the basis of the phenotypic and phylogenetic distinctiveness of our isolates, it is concluded that the strains represent a novel and distinct taxon at the genus level in a deeply branched lineage of the phylum Firmicutes. Significant phenotypic and genetic differences among strains JLT, JE and OL were not clearly found (Table 1). All their major physiological, chemotaxonomic and genetic properties (16S rRNA genes) were very similar to each other. Based on these findings, it seems justifiable that all three strains should be classified into one species. Hence, the name Tepidanaerobacter syntrophicus gen. nov., sp. nov. is proposed. The type strain is strain JLT.
Strains JLT, JE and OL were isolated from sludges of thermophilic anaerobic digesters in which high-strength organic solid wastes were digested. It is known that ethanol and lactate are important intermediates in the anaerobic degradation of organic compounds (such as carbohydrates) under thermophilic compounds (Stams & Zehnder, 1990). Therefore, the strains could play a role in the degradation of such compounds in syntrophic association with methanogens in the digestion process. Moreover, the strains could degrade monomeric and polymeric carbohydrates such as starch and pectin, suggesting that they could act as polysaccharide degraders in organic solid wastes in sludge.
Description of Tepidanaerobacter gen. nov.
Tepidanaerobacter (Te.pi.da.nae.ro.bac'ter. L. adj. tepidus moderately warm; Gr. pref. an not; Gr. n. aer air; N.L. masc. n. bacter rod; N.L. masc. n. Tepidanaerobacter moderately thermophilic anaerobic rod).
Gram-negative. Cells are non-motile, rod- or filament-shaped. Spores are not formed. Moderately thermophilic. Cells grow under strictly anaerobic conditions. Neither photosynthetic nor aerobic growth is observed. The main fatty acids are iso-C15 : 0, C16 : 0 and C15 : 1. Phylogenetic position is in a deeply branched lineage of the phylum Firmicutes. The type species is Tepidanaerobacter syntrophicus.
Description of Tepidanaerobacter syntrophicus sp. nov.
Tepidanaerobacter syntrophicus (syn.tro'phi.cus. Gr. pref. syn together with; Gr. adj. trophikos nursing, tending or feeding; N.L. masc. adj. syntrophicus pertaining to syntrophic substrate utilization).
Cells are irregular rods or sometimes filaments (1.5 to >10 µm long and 0.6–0.8 µm wide). Growth occurs between 25 and 60 °C with optimum growth at 45–50 °C. The pH range for growth is 5.5–8.5; optimum growth occurs at pH 7.0. Yeast extract is required for growth. In the presence of yeast extract, growth and substrate utilization can be observed with the following substrates: glucose, xylose, fructose, mannose, sucrose, starch and pectin. Some strains utilize the following substances: crotonate, ribose, malate, arabinose, galactose and raffinose. Growth and acid formation in pure culture are not observed with the following substrates in the presence of yeast extract: yeast extract itself, Casamino acids, H2/CO2 plus acetate, tryptone, betaine, pyruvate, lactate, glycerol, acetate, propionate, butyrate, succinate, fumarate, ethanol, methanol, 1-propanol, 1-butanol, benzoate, hydroquinone, phenol, 1,3-benzenediol or formate plus acetate. Thiosulphate is used as an electron acceptor. None of the following compounds are utilized as electron acceptors: sulphate, nitrate, sulphite, elemental sulphur or Fe(III) NTA. Hydrogenotrophic methanogens can also be used as the electron-accepting system. In the presence of hydrogenotrophic methanogens, ethanol, glycerol and lactate are used. None of the following substances are used in co-culture with methanogens: acetate, propionate, butyrate, methanol, 1-propanol, 1-butanol or benzoate. The G+C content of genomic DNA of the three known strains is 37–38 mol%.
The type strain is JLT (=JCM 12098T=NBRC 100060T=DSM 15584T), isolated from sludge of thermophilic digesters that decompose organic solid wastes. Strains JE (=JCM 12099=NBRC 100061=DSM 15585) and OL (=JCM 12100=NBRC 100062=DSM 15586) are reference strains.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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