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1 State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, PR China
2 Graduate School of the Chinese Academy of Sciences, Beijing 100080, PR China
Correspondence
Xiuzhu Dong
dongxz{at}sun.im.ac.cn
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Published online ahead of print on 14 January 2005 as DOI 10.1099/ijs.0.63565-0.
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of Syntrophobacter sulfatireducens TB8106T is AY651787.
A transmission electron micrograph of a cell of Syntrophobacter sulfatireducens TB8106T is available as supplementary material in IJSEM Online.
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). Propionate, one of the central intermediates in this process, is further degraded by the combined action of acetogenic bacteria and methanogenic archaea, due to the unfavourable energetics under standard thermodynamics. The methanogens make propionate oxidation energetically feasible by keeping the concentration of the products H2 and/or formate extremely low (Stams, 1994; Schink, 1997). So far, seven syntrophic propionate-oxidizing bacterial species have been described. The four mesophilic species form a cluster within the Gram-negative 
-subclass of the Proteobacteria, closely related to the mesophilic sulfate reducers. They are Syntrophobacter wolinii (Boone & Bryant, 1980; Wallrabenstein et al., 1994), Syntrophobacter fumaroxidans (Harmsen et al., 1998), Syntrophobacter pfennigii (Wallrabenstein et al., 1995) and Smithella propionica (Liu et al., 1999). Members of the genus Syntrophobacter can use sulfate as an electron acceptor and oxidize propionate via the methylmalonyl CoA pathway (Harmsen et al., 1993; Houwen et al., 1990; Plugge et al., 1993), whereas Smithella propionica, which grows axenically on crotonate, uses a six-carbon-intermediate pathway to oxidize propionate (Liu et al., 1999; de Bok et al., 2001). Unlike the mesophilic species, the three described thermophilic propionate-oxidizing species are all Gram-positive, spore-forming bacteria (Nilsen et al., 1996; Imachi et al., 2002; Plugge et al., 2001). In this study, we describe two novel mesophilic syntrophic propionate-oxidizing strains, TB8106T and WZH410. Phylogenetic analysis indicates that both strains are members of the genus Syntrophobacter. However, they are not closely related to strains of recognized Syntrophobacter species. Strains TB8106T and WZH410 were isolated from the granular sludge of two mesophilic upflow anaerobic sludge blanket (UASB) reactors, one treating brewery wastewater in Tsinghua University and the other treating bean curd wastewater in Wangzhihe bean curd manufactory (Beijing, China). Methanospirillum hungatei DSM 864T and Syntrophobacter fumaroxidans DSM 10017T were kindly provided by Dr Alfons Stams, Department of Microbiology, Wageningen University, The Netherlands.
A pre-reduced basal medium was used as described by Stams et al. (1992), and yeast extract (0·02 %, w/v) and tryptone (0·02 %, w/v) were added to stimulate growth. Routine cultivation was in anaerobic tubes (18x180 mm), sealed with butyl rubber stoppers. A gaseous atmosphere of N2/CO2 (80 : 20, 1·01x105 Pa) was used, except that H2/CO2 (80 : 20, 1·25x105 Pa) was used for cultivating methanogens. All inoculations and transfers were done with syringes and needles, and all cultures were incubated at 37 °C in the dark. For the isolation of co-cultures, direct serial dilution and the Hungate roll-tube technique (Hungate, 1969) were used, with 20 mM propionate as the sole carbon source. For the isolation of pure cultures of propionate-degrading bacteria, 20 mM propionate plus 20 mM sulfate were used. The purity of the strains in co-culture and pure culture was checked by microscopic examination, and by colony formation on solid media, as well as by the absence of growth in rich medium such as tryptone-peptone-yeast extract-glucose liquid medium (Holdeman et al., 1977).
Cell morphology was examined under a light microscope (Olympus BH-2) and an electron microscope (Hitachi H-600A). For electron microscopy studies, cells were negatively stained with uranyl acetate.
In order to determine the optimum growth conditions for strains TB8106T and WZH410, the basal medium with 20 mM propionate plus 20 mM sulfate and a 10 % inoculum were used. Growth at various pH values was determined in media adjusted with 1 M HCl or NaOH. Temperature profiles were determined by using a water bath (Guangming Medical Instrument Plant, Beijing) at 15 to 55 °C, at 1 °C intervals (pH 7·5). To determine the NaCl tolerance, 0–1000 mM NaCl was added to the medium. The generation time of the strains was determined by monitoring the OD600 of the culture at 37 °C at 1-day intervals for up to 60 days. Growth under the various conditions given above was determined by monitoring the OD600 after cultivation for up to 60 days.
To determine the substrate range of the strains in pure culture and in co-culture with methanogens, sterilized substrates were added to the basal medium to a final concentration of 20 mM (unless indicated otherwise), instead of propionate. To determine the electron acceptors for propionate degradation of the methanogen-free culture, each of the tested compounds (20 mM) was added to the propionate medium. After inoculation, the cultures were incubated for up to 90 days. Utilization of substrates by the strains was determined by monitoring the OD600 of the culture, substrate depletion and product formation. Fatty acids and CH4 were detected by using a gas chromatograph (GC-14B; Shimadzu), as described previously (Chen & Dong, 2004). All tests were performed in duplicate with a 10 % inoculum.
Genomic DNA was extracted and purified from strains TB8106T and WZH410 grown on propionate plus sulfate, as described by Marmur (1961). The mol% G+C content of DNA was determined by the thermal denaturation method (Marmur & Doty, 1962), by using a DU800 spectrophotometer (Beckman) with Escherichia coli K-12 as the reference. The 16S rRNA gene was amplified and sequenced according to Chen & Dong (2004). Sequencing was performed by Sangon Biological Engineering Technology Service, Shanghai, China, using ABI PRISM Big Dye Terminator cycle sequencing ready reaction kits (Perkin Elmer) and an ABI PRISM 377XL DNA sequencer. The 16S rRNA gene sequence of strain TB8106T was submitted to GenBank and EMBL to search for similar sequences using the BLAST algorithm. The best matching sequences were retrieved from the database and aligned, and similarity analysis was performed using the CLUSTAL_X program (Thompson et al., 1997). Phylogenetic trees were constructed by using the neighbour-joining, maximum-likelihood and maximum-parsimony methods, implemented in the MEGA 2 program (Kumar et al., 2001) and the PHYLIP package (Felsenstein, 1993). The topologies of the resultant tree were evaluated by bootstrap analysis (Felsenstein, 1985), based on 1000 resamplings.
DNA–DNA hybridization was performed at 73 °C on the basis of DNA–DNA liquid reassociation rate (De Ley et al., 1970), using a DU800 spectrophotometer (Beckman).
Propionate-degrading methanogenic enrichments were developed from anaerobic digester sludge by diluting the sludge at a ratio of 1 : 10 with the basal medium plus 20 mM sodium propionate. After 3 weeks of incubation at 37 °C, the enriched cultures degraded propionate and produced large amounts of CH4. The enrichments were subcultured six times, and then the roll-tube technique was applied with propionate-containing agar medium. After incubation for 10 weeks, colonies were visible in the roll tubes. Large colonies at dilutions of 10–4 and 10–5 that emitted green fluorescence at a wavelength of 420 nm were picked and inoculated into the propionate medium. This roll-tube procedure was repeated several times over a period of 2 years, until two co-cultures that produced methane from propionate were obtained. The co-cultures consisted of two kinds of cell with different morphologies: one was egg-shaped and the other resembled Methanobacterium formicicum cells. Colonies of the co-cultures were yellowish, circular and 1·5–2·0 mm in diameter, and emitted green fluorescence at 420 nm after 10 weeks of incubation. The propionate oxidizers were further purified by inoculating the co-culture onto medium containing 20 mM propionate plus 20 mM sulfate; 10 mM 2-bromoethane sulfonate was also added to inhibit the methanogen. After being subcultured 12 times on this medium, monocultures designated as strains TB8106T and WZH410 were obtained. Hardly any growth of the strains as a pure culture was observed on solid medium, and propionate could not be degraded in the absence of sulfate, whereas propionate was degraded to acetate and CH4 in an artificially constructed co-culture with Methanospirillum hungatei DSM 864T after 50–60 days.
Cells of strains TB8106T and WZH410 were non-motile, Gram-negative, egg-shaped, and 1·0–1·3x1·8–2·2 µm in size (see Supplementary Figure in IJSEM Online). Spore formation was never observed. The strains were strictly anaerobic; no growth occurred when exposed to air. The temperature range for growth was 20–48 °C, with optimum growth occurring at 37 °C. The pH range for growth was 6·2–8·8, with optimum growth occurring at pH 7·0–7·6. NaCl was tolerated up to a concentration of 100 mM (best growth occurred at concentrations below 50 mM). Addition of 0·02 % yeast extract and tryptone stimulated, but was not necessary for, growth. In pure culture on propionate plus sulfate, 20 mM propionate was oxidized to 20 mM acetate within 12–15 days. The doubling time of strain TB8106T was 52–55 h at 37 °C, and the growth rate was approximately 0·12 day–1. The growth yield of strain TB8106T on propionate plus sulfate was 3·3 g dry weight (mol propionate)–1.
Sulfate and thiosulfate were utilized as electron acceptors for propionate oxidation, but not sulfur, nitrate or fumarate. Strain TB8106T utilized sulfite, whereas WZH410 did not. Both strains fermented pyruvate in pure culture. The following substrates were not used in either pure or co-culture: H2/CO2 (80 : 20, v/v), formate, H2 plus fumarate, formate plus fumarate, acetate, butyrate, crotonate, succinate, fumarate, oxaloacetate, malate, citrate, malonic acid, glutarate (10 mM), alanine (10 mM), glycine (10 mM), asparagine (10 mM), benzoate (10 mM), methanol, ethanol, 1-propanol, 1-butanol, glucose (10 mM), fructose (10 mM), xylose (10 mM) and arabinose (10 mM). Propionate was the sole substrate for the co-culture.
The G+C contents of the genomic DNA of strains TB8106T and WZH410 were 58·5 and 58·7 mol%, respectively. The partial 16S rRNA gene sequence (500 bp) similarity between strains TB8106T and WZH410 was 99·94 %, indicating that they belonged to the same species.
To ascertain the phylogenetic position of strain TB8106T, the complete 16S rRNA gene sequence (1553 bp) was compared with the most similar sequences retrieved from GenBank. On the basis of a consensus 1361 bp of the 16S rRNA gene sequence, a phylogenetic tree (Fig. 1), rooted with E. coli ATCC 11775T, was constructed. The tree clearly showed that strain TB8106T clustered within the genus Syntrophobacter, in the -subclass of the Proteobacteria, and was most closely related to strain HP1.1 (Zellner et al., 1996) (99·2 % similarity), a highly purified propionate-degrading culture, and Syntrophobacter fumaroxidans DSM 10017T (94·7 % similarity) and Syntrophobacter pfennigii DSM 10092T (94·6 % similarity).
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). In monoculture, Syntrophobacter wolinii and Syntrophobacter fumaroxidans both fermented fumarate, and Syntrophobacter fumaroxidans also utilized fumarate as an electron acceptor for propionate oxidation. Syntrophobacter pfennigii utilized lactate not only in monoculture, but also in methanogenic co-culture. All these features were not shared by strains TB8106T and WZH410. Furthermore, strains TB8106T and WZH410 had obviously higher growth rates on propionate plus sulfate (0·12 day–1) than Syntrophobacter fumaroxidans DSM 10017T (0·024 day–1). In addition, the novel isolates grew well at 48 °C and pH 8·8, a higher growth temperature and pH than those for other members of the genus Syntrophobacter. DNA–DNA hybridization between strain TB8106T and Syntrophobacter fumaroxidans DSM 10017T showed a low genome DNA–DNA relatedness (12·5 %), indicating that they belong to different species.
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The description of the genus Syntrophobacter of Boone & Bryant (1980) indicated that sulfate did not serve as an electron acceptor in the energy metabolism. However, the later-described syntrophic propionate-degrading species Syntrophobacter fumaroxidans, Syntrophobacter pfennigii and the novel isolates in this work were all demonstrated to be capable of coupling propionate oxidation to sulfate reduction. In addition, Syntrophobacter wolinii was also shown to be able to use sulfate as an electron acceptor (Wallrabenstein et al., 1994). Hence, it is necessary to emend the description of the genus to emphasize the sulfate reduction capability, in accordance with its close phylogenetic relationship with sulfate reducers.
Emended description of the genus Syntrophobacter
The description is based on the phenotypic data of Boone & Bryant (1980), Wallrabenstein et al. (1994, 1995) and Harmsen et al. (1998).
Gram-negative, non-motile, rod- to egg-shaped cells. Endospores are not formed. Strictly anaerobic chemo-organotrophs. Mesophilic. Growth by fermentation or by reduction of sulfate to sulfide. Pure cultures oxidize propionate with sulfate as the electron acceptor. Propionate is fermented syntrophically to acetate and CO2 in the presence of hydrogen- and/or formate-utilizing methanogens (e.g. Methanospirillum hungatei). The G+C content of the genomic DNA is 56·7–60·6 mol%.
The type species is Syntrophobacter wolinii.
Description of Syntrophobacter sulfatireducens sp. nov.
Syntrophobacter sulfatireducens (sul'fa'ti.re.du'cens. N.L. n. sulfas -atis, sulfate; L. v. reducere to lead back, bring back; N.L. part. adj. sulfatireducens reducing sulfate).
Morphology and general characteristics are as described for the genus. Egg-shaped cells, 1·0–1·3x1·8–2·2 µm in size, single, in pairs or in chains. Pure cultures oxidize propionate and pyruvate to acetate, with sulfate as electron acceptor; pyruvate is also oxidized by fermentation. Does not grow on H2/CO2, formate, H2 plus fumarate, formate plus fumarate, acetate, butyrate, crotonate, succinate, fumarate, oxaloacetate, malate, citrate, malonic acid, glutarate, alanine, glycine, asparagine, benzoate, methanol, ethanol, 1-propanol or 1-butanol. Sulfate and thiosulfate are both used as electron acceptors, but sulfur, nitrate and fumarate are not reduced. Growth is optimal at pH 7·0–7·6 and at 37 °C. The G+C content of genomic DNA is 58·5–58·7 mol%. Habitat is granular sludge of UASB reactors.
The type strain is TB8106T (=AS 1.5016T=DSM 16706T), which was isolated from a UASB reactor treating brewery wastewater, in Beijing, China.
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ACKNOWLEDGEMENTS |
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