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1 Department of Microbiology, University of Georgia, Athens, GA 30602, USA
2 Department of Geology, University of Georgia, Athens, GA 30602, USA
3 Savannah River Ecology Laboratory, Aiken, SC 29802, USA
Correspondence
Juergen Wiegel
jwiegel{at}uga.edu
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ABSTRACT |
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). A few anaerobic micro-organisms have been isolated and characterized from low pH environments, including methanogens (Williams & Crawford, 1985; Horn et al., 2003) and clostridia (Hamman & Ottow, 1976; Kuhner et al., 2000). Since members of the genus Clostridium are metabolically versatile and capable of forming endospores, they are ubiquitous and can be found in various extreme environments such as the deep-sea, hot springs and the intestinal tracts of animals.
In this paper, we report the characterization of an anaerobic, acid-tolerant, spore-forming bacterium which falls into the radius of the genus Clostridium sensu stricto (Wiegel et al., 2005), formerly known as Collins' cluster I and II of the genus Clostridium (Collins et al., 1994).
A constructed wetland system was built to treat acid sulfate water at the US Department of Energy's Savannah River Site near Aiken, SC, USA. The acid sulfate water (pH
2.0) was introduced at the top of each cylindrical cell in the system, containing sand mixed with horse manure, wood chips and limestone. Within a few months of operation, the sediment developed a series of different reaction zones that were distinguishable based on a variety of physico-chemical attributes (Thomas, 2003; Lee, 2005). Sediment samples for the enrichments were taken from the top layer of the core where conditions were relatively oxidized, the pH was around 3.5 and the sediment was dominated by iron oxyhydroxide.
Enrichments under anaerobic and acidic conditions (pH 3.5, 4.5 and 5.5) were prepared using a medium designed for sulfate-reducing bacteria (Widdel & Bak, 1992) using a modified Hungate technique (Ljungdahl & Wiegel, 1986). The medium contained either acetate (15 mM) or lactate (15 mM) as the sole carbon source and energy source supplemented with 0.1 % yeast extract. The enrichments were incubated at 37 °C for up to one month.
A pure isolate, designated strain JW/YJL-B3T, was obtained from the enrichments by three subsequent rounds of single colony isolation using the agar (1.5 % w/v)-shake-roll-tube technique (Ljungdahl & Wiegel, 1986). Colonies of the isolate appeared after 1–2 days and were irregular, mostly translucent, filamentous and less than 1.5 mm in diameter.
For the phylogenetic analysis, DNA was extracted from the isolate as described previously (Lee et al., 2005) and amplified with a bacterial domain-specific primer set for the 16S rRNA gene, 27 forward and 1492 reverse (Lane, 1991). The PCR amplification was carried out with initial denaturation at 94 °C for 2 min and followed by 10 cycles of denaturation (94 °C, 30 s), annealing (58 °C, 30 s) and extension (72 °C, 1 min), 10 cycles of denaturation (94 °C, 30 s), annealing (58 °C, 45 s) and extension (72 °C, 1 min 15 s) and 10 cycles of denaturation (94 °C, 30 s), annealing (58 °C, 1 min) and extension (72 °C, 1 min 30 s). Final extension was for 7 min at 72 °C. PCR products were purified using a QIAquick PCR purification kit (Qiagen) and sequenced by Macrogen Inc. (Seoul, Korea). Retrieved 16S rRNA gene sequences were analysed using BLAST and aligned manually against sequences obtained from the genbank database using CLUSTAL_X v1.81 (Thompson et al., 1997) and GeneDoc v2.6.02 (www.psc.edu/biomed/genedoc). Phylogenetic trees were constructed by the neighbour-joining method (Saitou & Nei, 1987) and FITCH (Fitch & Margoliash, 1967) using the Jukes and Cantor model (Jukes & Cantor, 1969) with the PHYLIP v3.6a2.1 phylogenetic analysis package (Felsenstein, 2001).
A nearly complete 16S rRNA gene sequence was obtained for strain JW/YJL-B3T, comprising of 1391 bp [approximate positions 47–1467 according to the Escherichia coli (GenBank accession number X80725) numbering scheme]. Based on 16S rRNA gene sequence similarity, strain JW/YJL-B3T fell into the radius of Clostridium sensu stricto (Wiegel et al., 2005). The closest relative was Clostridium drakei (96.2 % gene sequence similarity) (Fig. 1).
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Morphology was studied using light microscopy (VANOX phase-contrast microscope; Olympus) and transmission electron microscopy (100CX; JEOL). Vegetative cells grown in phosphate-buffered medium were straight rods, occurring singly or in pairs. The cells were 0.5–1.0 µm in diameter and 3.0–9.0 µm in length. Endospores detected in the late exponential growth phase were subterminal and oval in shape without swelling of the cells. Cells of strain JW/YJL-B3T had peritrichous flagella (Fig. 2b), but motility was not observed during light microscopy. Cells stained Gram-negative at all growth phases (Doetsch, 1981), while electron microscopy and phylogenetic position indicated a Gram-type positive cell-wall structure (Fig. 2c) as expected from the 16S rRNA gene sequence–based phylogeny. Thus, the novel strain is Gram-stain negative but is Gram-type positive (Wiegel, 1981).
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) range for growth was determined using media buffered with 10 mM each of MES, HEPES and TAPS in combination with 2 mM phosphate. The pH25 °C range for growth was pH 3.8–8.9, with an optimum at pH 7.0–7.5. Although the optimum pH was in the neutral range, the strain grew well under mildly acidic conditions, i.e., growth at pH 4.5–5.0 occurred at 52 % of the optimal growth rate. Growth was not detected at or below pH 3.5 or at or above pH 9.2. The salinity range for growth was obtained using phosphate-buffered medium supplemented with various concentrations of NaCl and KCl (9 : 1). The isolate grew optimally in the absence of NaCl and KCl, but showed growth up to 1.5 % (w/v) salinity. No growth was detected at 2 % salinity. The doubling time at 37 °C and pH25 °C 6.5 with 0.3 % glucose was 1.7 h. Detailed results of other morphological and physiological characteristics are summarized and compared with closely related species in Table 1.
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) and lipase. Gelatin was hydrolysed, but not casein. Haemolysis was detected on blood agar. The utilization of various carbon sources was studied using phosphate-buffered basal medium supplemented with 0.02 % yeast extract and the test substrate. Strain JW/YJL-B3T used beef extract, Casamino acids, peptone, tryptone, cellobiose, fructose, galactose, glucose, lactose, maltose, mannose, raffinose, ribose, sucrose, xylose, pyruvate, glutamate and inulin as carbon and energy sources. Acetate, lactate, arabinose, trehalose, inositol, mannitol, sorbitol, xylitol and cellulose did not support growth of the strain. There was no indication of autotrophic growth under an atmosphere of 15 p.s.i. of CO2 and H2 (80 : 20). The strain grew in classical peptone–sugar media including peptone–yeast extract (PY), peptone–yeast extract–glucose (PYG), reinforced clostridial medium (RCM, Difco) and thioglycolate broth (Difco). The fermentation end products from glucose (20 mM) were acetate, butyrate and ethanol. The potential use of electron acceptors was determined in cultures incubated for at least 2 weeks using phosphate-buffered basal medium containing 0.2 % Casamino acids as an electron donor and an inoculum of 3 % (v/v) of an exponentially growing culture. The use of electron acceptors was determined by measuring growth (OD600), sulfide, ammonium or nitrite production or colour-change. However, none of the following electron acceptors tested at 20 mM was utilized: fumarate, nitrate, nitrite, sulfate, thiosulfate, elemental sulfur, Fe(III), 9,10-anthraquinone 2,6-disulfonate (AQDS), Mn(IV) or sulfite at 2 mM. Oxygen was not used as a terminal electron acceptor when tested in a 150 ml serum bottle laid horizontally containing 5 ml culture (basal medium) and air as head gas.
Strain JW/YJL-B3T was resistant only to tetracycline at 10 µM and sensitive to 10 µM ampicillin, chloramphenicol, erythromycin, rifampicin and streptomycin and 100 µM tetracycline. The G+C content of genomic DNA, determined by the HPLC method as described previously (Mesbah et al., 1989; Lee et al., 2005), was 30.8 mol% (mean of 4 replicates of the nuclease digest and HPLC runs).
Based on morphological, physiological and phylogenetic characteristics, we propose to place strain JW/YJL-B3T as the type strain of a novel taxon, Clostridium aciditolerans, sp. nov., belonging to the genus Clostridium sensu stricto within the family Clostridiaceae (Garrity et al., 2004; Wiegel et al., 2005).
Description of Clostridium aciditolerans sp. nov.
Clostridium aciditolerans (a.ci.di.to'le.rans. N.L. n. acidum an acid; L. part. adj. tolerans tolerating; N.L. part. adj. aciditolerans acid-tolerating).
Cells are straight to slightly curved rods, 0.5–1 µm in diameter and 3.0–9.0 µm in length. Spores are subterminal and oval in shape and do not swell the cell. Retarded peritrichous flagellation is observed. Although the type strain stains Gram-negative at all growth phases, the strain is Gram-type positive. The temperature range for growth is 20–45 °C, with an optimum around 35 °C. No growth is observed at or below 18 °C or at or above 47 °C. The pH25 °C range for growth is from pH 3.8 to 8.9, with an optimum at pH 7.0–7.5. Growth at pH 4.5–5.0 takes place at 52 % of the optimal growth rate; no growth is observed at or below pH 3.5 or at or above pH 9.2. The salinity range for growth is from 0 to 1.5 % NaCl (w/v). In the presence of 0.02 % yeast extract, the following substrates serve as carbon and energy source: beef extract, Casamino acids, peptone, tryptone, cellobiose, fructose, galactose, glucose, lactose, maltose, mannose, raffinose, ribose, sucrose, xylose, pyruvate, glutamate and inulin. Fe(III), nitrate, thiosulfate, elemental sulfur, sulfate, sulfite, MnO4 and fumarate are not used as electron acceptors. The main organic fermentation end-products from glucose are acetate, butyrate and ethanol. The strain is resistant to tetracycline (10 µM) and sensitive to ampicillin (10 µM) chloramphenicol (10 µM), erythromycin (10 µM), rifampicin (10 µM) and streptomycin (10 µM). The G+C content of the genomic DNA is 30.8 mol% (HPLC).
The type strain, JW/YJL-B3T (=DSM 17425T=ATCC BAA-1220T), was isolated from a sediment sample from a constructed wetland system receiving acid sulfate water.
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ACKNOWLEDGEMENTS |
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