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School of Municipal and Environmental Engineering, Harbin Institute of Technology, Harbin 150090, PR China
Correspondence
Nanqi Ren
rnq{at}hit.edu.cn or
ixdf{at}yahoo.com.cn
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ABSTRACT |
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Published online ahead of print on 25 November 2005 as DOI 10.1099/ijs.0.63926-0.
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strains YUAN-3T and X-29 are AY295777 and AY833421, respectively.
Micrographs of colonies and cells of strain YUAN-3T are available as supplementary material in IJSEM Online.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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). During a survey of the microbial community in a hydrogen-producing bioreactor, two obligately anaerobic, autoaggregative, non-spore-forming bacterial strains were isolated from anaerobic sludge of molasses wastewater in a continuous stirred-tank reactor (CSTR). The strains produced H2, CO2, ethanol and acetic acid from glucose fermentation. Phylogenetically, the strains were affiliated with the Clostridium leptum rRNA subgroup (Collins et al., 1994); however, they were only distantly related to any existing genera in this cluster. Based on the distinctive phenotypic, genomic and phylogenetic characteristics of these two strains, it is proposed that they represent a novel species in a new genus.
Strains YUAN-3T and X-29 were isolated in pre-reduced peptone/yeast extract/glucose (PYG) medium (Holdeman et al., 1977) by serial dilution and the Hungate roll-tube technique (Hungate, 1969). Single colonies were picked and transferred to the same broth and incubated at 35 °C for 2 days. The roll-tube procedure was repeated several times until a pure culture was obtained. Routine cultivation was in PYG broth in anaerobic tubes (18x180 mm) sealed with butyl rubber stoppers under a gaseous atmosphere of 100 % N2 (200 kPa) at 35 °C.
Morphology and life cycle were examined in cultures grown on PYG medium using a light microscope (Olympus BX-51) and an EM (FEI TECNI G2). For EM studies, bacterial cells grown in PYG at 35 °C for 2 days were stained with phosphotungstic acid (3 %). For ultrathin section examination of the cell wall, bacterial cells were fixed with osmic acid and embedded in araldite; samples were then sliced and stained with lead citrate (Reynolds, 1963).
Gases were measured by GC (GC-SC2) equipped with a thermal conductivity detector and a 2·0 m stainless steel column packed with TDS-01 (60/80 mesh). N2 was used as carrier gas at a flow rate of 70 ml min–1. Fatty acids and alcohols were detected by GC (GC-122) equipped with a hydrogen flame-ionization detector and a 2·0 m stainless steel column packed with GDX103 (60/80 mesh). For measurement of non-volatile fatty acids, methyl esters were derived from samples according to Holdeman et al. (1977) before being analysed. Column temperatures were 220, 190 and 190 °C for the measurement of volatile fatty acids, alcohols and non-volatile fatty acids, respectively. N2 was used as carrier gas at a flow rate of 60 ml min–1.
Generation time of the strains was determined by monitoring OD600 of the PYG culture at 35 °C at 2 h intervals up to 48 h. Temperature profiles were determined in PYG broth using a water bath at temperatures of 15–55 °C at intervals of 1 °C. The pH range for growth was determined for the culture in PYG broth at various pH values adjusted with HCl or NaOH (1 mol l–1). Growth was determined by measuring the OD600 of cultures at 1, 3 and 7 days. Biochemical traits were determined using both conventional methods and the API 50CH system (bioMérieux). Enzyme activities were analysed using both conventional methods and the API ZYM system (bioMérieux). All tests were performed in duplicate.
For extraction of cell walls, crude cells were disrupted by sonication, separated from unbroken cells by fractional centrifugation and purified using trypsin and 2 % SDS as reported previously (Evtushenko et al., 2000). The diagnostic isomers of diaminopimelic acid (DAP) were detected by the TLC method (Lechevalier & Lechevalier, 1980). Cellular fatty acids were extracted, methylated and analysed using the standard MIDI (Microbial Identification) system (Miller, 1982; Sasser, 1990).
Genomic DNA was extracted and purified using the bacterial DNA mini kit (Watson Biotechnologies) according to the manufacturer's instructions. The G+C content of the DNA was determined by the thermal denaturation method (Marmur & Doty, 1962) using a DU800 spectrophotometer with Escherichia coli K-12 as the reference. The 16S rRNA gene was amplified by PCR using a pair of universal primers, 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1541R (5'-AAGGAGGTGATCCAGCC-3'), corresponding to base positions 8–27 and 1525–1541, respectively, of the 16S rRNA gene of Escherichia coli (Winker & Woese, 1991). The genomic DNA extracted was used as a template and PCR amplification was performed with the GenAmp PCR System 9700. PCR products were detected by agarose gel electrophoresis and visualized by UV fluorescence after ethidium bromide staining. PCR products were purified using a UNIQ-10 PCR product purification kit (Sangon) and cloned in Escherichia coli JM109 using the pGEM-T plasmid vector system (Promega) as recommended by the manufacturer. Primers T7 (5'-GTAATACGACTCACTATAGG-3') and M13R (5'-CAGGAAACAGCTATGACCAT-3') were used for sequencing the 16S rRNA gene fragment. Sequencing was performed by Bioasia Biological 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 YUAN-3T 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; similarity analysis was performed using the program CLUSTAL_X (Thompson et al., 1997). Phylogenetic trees were constructed from the evolutionary distance matrix calculated using the neighbour-joining method (Saitou & Nei, 1987) with Kimura's two-parameter method (Kimura, 1980). Neighbour-joining analysis was performed with the program MEGA3 (Kumar et al., 2004). Robustness of the resultant tree topology was evaluated by bootstrap resampling analysis (Felsenstein, 1985) with 1000 replicates.
Colonies of strains YUAN-3T and X-29 on PYG agar were milk-white, circular, opaque and slightly concave with an obvious ring margin, reaching 1·5–3·0 mm in diameter after cultivation at 35 °C for 48 h. Cells were Gram-positive, non-spore-forming rods (0·4–0·8x1·5–8·0 µm), motile by means of peritrichous flagella. Strain YUAN-3T was autoaggregative and autofluorescent, whereas strain X-29 showed non-aggregative and non-fluorescent growth. The young culture (12–24 h) consisted of rods or filaments (0·4–0·8x4·0–8·0 µm), occurring singly or in pairs; strain YUAN-3T began to form autoaggregative granules in shake cultures. In 3- to 4-day-old cultures, the rods and filaments fragmented into shorter elements (about 1·5–4·0 µm). In older cultures, regular rods predominated and occurred singly or in pairs; strain YUAN-3T formed large autoaggregative granules (generally 0·5–5·0 mm, but sometimes up to 1·5 cm) in shake cultures and the culture medium was clear. However, cultures of strain X-29 were non-aggregative and homogeneous. Polysaccharide capsules (0·1–0·4 µm) were usually observed on agar media and in liquid culture; those of strain YUAN-3T were usually thicker than those of strain X-29. A Gram-positive cell wall and cell inclusion bodies (polyphosphate and poly-
-hydroxybutyrate) were confirmed by ultrathin section EM of strain YUAN-3T (available as supplementary material in IJSEM Online).
Strains YUAN-3T and X-29 grew in pre-reduced media and growth was completely inhibited by air. Any one of yeast extract, tryptone or peptone could be used as a nitrogen source by both strains but, apart from (NH4)2HPO4, inorganic nitrogen compounds such as NH4Cl, NH4NO3, (NH4)2SO4 and KNO3 could not be used. Both strains grew at 20–44 °C and pH 3·5–9·0, with optimum growth at 35 °C and approximately pH 4·5–5·0. The strains could grow in the presence of 0–2 % (w/v) NaCl. The mean generation time of the two strains was 4·2 h when grown in PYG broth at 35 °C. The growth yield of strain YUAN-3T in PYG broth was 1·205 g cell dry mass (l culture)–1.
Apart from the fact that strain YUAN-3T strongly autoaggregated, whereas strain X-29 did not, the two strains exhibited almost identical physiological and biochemical characteristics as determined using routine methods and the API 50CH and API ZYM systems. Both isolates hydrolysed gelatin, curdled milk and produced acid from a few sugars such as raffinose and sucrose (detailed results are listed in the species description). YUAN-3T did not produce acid from mannitol, but X-29 did. YUAN-3T weakly hydrolysed starch and cellulose, but X-29 did not. Strain YUAN-3T was positive for leucine arylamidase, whereas strain X-29 was negative. The products of glucose fermentation by strains YUAN-3T and X-29 were acetate, ethanol, H2 and CO2.
The cell wall hydrolysates of the two strains were rich in LL-DAP. The cellular fatty acids of strain YUAN-3T were characterized mainly by iso-branched fatty acids, predominantly iso-C16 : 0 (44·18 %) and iso-C12 : 0 (26·67 %); anteiso-C17 : 0 (5·25 %) and C16 : 0 (4·98 %) fatty acids were also relatively abundant. The cellular fatty acid compositions were obviously different from those of phylogenetically related Clostridium species; most of the mesophilic members are characterized by a higher percentage of unsaturated fatty acids and the absence of branched-chain fatty acids (Kaneda, 1991).
Similarity between the complete 16S rRNA gene sequences of strains YUAN-3T and X-29 was 100 % and their DNA G+C contents were 47·8 and 49·0 mol%, respectively. All the results above indicate the single species status of the two isolates.
To determine the phylogenetic position of the isolates, the complete 16S rRNA gene sequences (1506 bp) were compared with the most similar sequences and those of representatives of the Clostridiaceae retrieved from GenBank. On the basis of a consensus 1334 bp sequence of the 16S rRNA gene, a phylogenetic tree, rooted with Acidaminobacter hydrogenoformans DSM 2784T, was constructed (Fig. 1). Phylogenetic analysis showed that the strains belonged to the C. leptum rRNA subgroup (Collins et al., 1994). This group consists of phenotypically and phylogenetically diverse groups (Chen & Dong, 2004), including spore-forming Clostridium species (Clostridium sporosphaeroides, C. leptum and C. cellulosi), Ruminococcus species (Ruminococcus albus, R. flavefaciens, R. bromii and R. callidus), Anaerofilum species (Anaerofilum agile and A. pentosovorans) (Zellner et al., 1996), Eubacterium species, Faecalibacterium prausnitzii (Duncan et al., 2002) and Papillibacter cinnamivorans (Defnoun et al., 2000). Strain YUAN-3T showed the highest 16S rRNA gene sequence similarity (91·8 %) to C. cellulosi; similarities were 84·0–88·8 % with other related species in the C. leptum subgroup and lower than 86·0 % with other representatives of the Clostridiaceae. The large sequence divergence indicated that the isolates represent a new genus in this cluster.
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), enabled them to be distinguished from related Clostridium species. They differed from Ruminococcus species in their rod shape, different biochemical traits and DNA G+C content. They differed from Eubacterium siraeum in glucose fermentation and other features shown in Table 1. They produced a large amount of hydrogen, enabling them to be distinguished from Anaerofilum species. F. prausnitzii has a Gram-negative cell wall and produces butyrate, D-lactate and formate but no hydrogen from glucose fermentation, enabling it to be differentiated from the two isolates. Unlike P. cinnamivorans, they were motile by peritrichous flagella.
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Description of Ethanoligenens gen. nov.
Ethanoligenens [E.tha.no.li.gen'ens. N.L. n. ethanol -is ethanol; L. part. adj. genens (from L. v. genere to produce) producing; N.L. neut. n. Ethanoligenens ethanol-producing (bacterium)].
Gram-positive, motile, non-spore-forming, straight rods. Obligately anaerobic. Oxidase- and catalase-negative. Cell wall peptidoglycan contains LL-DAP. Cellular fatty acids consist mainly of iso-branched fatty acids, predominantly iso-C16 : 0 and iso-C12 : 0. Mesophilic. Grow at 20–44 °C and pH 3·5–9·0. Chemo-organotrophic. Amino acids and peptides may serve as nitrogen sources. Various mono-, di- and oligosaccharides are fermented. Gelatin and aesculin are hydrolysed. The major fermentation products from glucose include ethanol, acetate, hydrogen and carbon dioxide; propionate and succinate are not produced. Sulfate is not reduced. The G+C contents of the genomic DNA of the known strains are 47·8 and 49·0 mol%. Belongs to the C. leptum rRNA subgroup. Only one species, Ethanoligenens harbinense, is described so far; this species has been designated the type species.
Description of Ethanoligenens harbinense sp. nov.
Ethanoligenens harbinense (har.bin'en.se. N.L. neut. adj. harbinense from Harbin, where the type strain was isolated).
General morphological, chemotaxonomic and growth characteristics are as described for the genus. Cells are 0·4–0·8x1·5–8·0 µm. Colonies on PYG agar are milk-white, circular, opaque, slightly concave with an obvious ring margin, reaching 1·5–3·0 mm in diameter after cultivation at 35 °C for 48 h. Optimal growth occurs at 35 °C. The pH range for growth is 3·5–9·0, with optimum growth at pH 4·5–5·0. Acid is produced from a few sugars, including D-glucose, D-fructose, D-galactose, cellobiose, D-maltose, sucrose, raffinose, ribose, trehalose, D-lactose, D-turanose, mannose, melibiose and salicin. Acid is not produced from arabinose, inulin, sorbose, rhamnose, glycogen, adonitol, dulcitol, erythritol, inositol, sorbitol, melezitose, xylitol, D-lyxose, D-tagatose, arabitol or fucose. No acid is produced from the following compounds: methanol, ethanol, 1-propanol, pyruvate, fumarate, malate, succinate, malonic acid, hippurate, -hydroxybutyric acid, phenylacetic acid or xylan. Curdles milk. Urease is produced. Arginine dihydrolase, lecithinase and lipase are not produced. Positive for esterase (C4), esterase lipase (C8), lipase (C14), valine arylamidase, trypsin, 
-chymotrypsin, -glucuronidase, N-acetyl--glucosaminidase, -mannosidase and -fucosidase. Negative for alkaline phosphatase, cystine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase, -galactosidase, -galactosidase and -glucosidase. Positive for indole, methyl red and Voges–Proskauer tests. Does not use sulfate or nitrate as electron acceptor. No H2S is produced from peptone or thiosulfate. The major cellular fatty acids are iso-C16 : 0 (44·18 %), iso-C12 : 0 (26·67 %), anteiso-C17 : 0 (5·25 %) and C16 : 0 (4·98 %).
The type strain is YUAN-3T (=JCM 12961T=CGMCC 1.5033T), isolated from the anaerobic sludge of molasses wastewater in a CSTR.
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ACKNOWLEDGEMENTS |
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