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Department of Biotechnology, Center for Chemistry and Chemical Engineering, Lund University, PO Box 124, SE-221 00 Lund, Sweden
Correspondence
Dores G. Cirne
Dores.Cirne{at}biotek.lu.se
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain R1T is AY858804.
Present address: PROIMI-CONICET, Av. Belgrano y Pasaje, Caseros, 4000 Tucumán, Argentina. 
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). Not many anaerobic lipolytic micro-organisms are known. Most of the information available on anaerobic lipase production is related to rumen. However, the number of described lipid-hydrolysing bacteria occupying the lipid-hydrolysing ecological niche in this ecosystem is very small with a few obligately anaerobic rumen bacteria described, Anaerovibrio lipolytica, Butyrivibrio fibrisolvens strain S2, isolate LIP4, which cluster in the genus Propionibacterium, and LIP5, which is a member of the clostridial cluster XIVa (Jarvis et al., 1998). Jarvis et al. (1999) isolated a glycerol-fermenting clostridial strain, LIP1, that possesses lipolytic activity. This strain is also a member of the clostridial cluster XIVa. Dighe et al. (1998) reported the isolation of a novel anaerobic lipolytic species, Selenomonas lipolytica, from an anaerobic lagoon receiving wastewater from an edible-oil mill. Svetlitshnyi et al. (1996) described a novel species, Thermosyntropha lipolytica, which is alkalitolerant and thermophilic.
In the genus Clostridium, whose members exhibit a wide range of phenotypic characteristics (Van Dyke & McCarthy, 2002), to the authors' knowledge, only 10 members of the genus have been reported to produce lipase, Clostridium botulinum, C. aurantibutyricum, C. novyi, C. sporogens (Dighe et al., 1998), C. tetanomorphum, C. tetani (Wilde et al., 1989, 1997), C. ghonii (Sneath, 1986), C. aerotolerans (van Gylswyk & van der Toorn, 1987), isolate LIP5 (Jarvis et al., 1998) and isolate LIP1 (Jarvis et al., 1999). However, little has been reported about their actual lipolytic activity. All of these micro-organisms, except isolates LIP1 and LIP5, are included in cluster I of the genus Clostridium, which is known for exhibiting high levels of intracluster similarity, despite having markedly different phenotypes (Collins et al., 1994).
In this paper, phylogenetic and phenotypic characterization of a novel anaerobic lipolytic micro-organism is described. Strain R1T was isolated from bovine rumen content collected from a slaughterhouse located near Lund, Sweden.
The liquid medium (denoted medium A) used in the enrichment and as a base medium, in most cultivations of R1T, contained the basal salts described by Markossian et al. (2000) supplemented with 0·1 % (w/v) yeast extract, 0·0025 % (w/v) rezasurin and 120 mg L-cysteine hydrochloride l–1. For isolation, 2 % (v/v) olive oil was added directly to each serum bottle as a lipid source. The anaerobic conditions were as described by Ljungdahl & Wiegel (1987). Lipolytic activity of isolate R1T was detected in rhodamine B medium consisting of medium A supplemented with 0·75 % (w/v) gum arabic, 2 % (v/v) olive oil and 0·001 % (w/v) rhodamine B (Jarvis & Thiele, 1997; Kouker & Jaeger, 1987). To obtain a solid medium, 2 % agar was added. The ability to grow under aerobic conditions was tested in rhodamine B medium. Tolerance to reducing agent was studied by inoculating medium A containing 500 mg L-cysteine hydrochloride l–1. All media had a final pH of 6·8–7·2 after autoclaving. Liquid cultures were grown with shaking (120 r.p.m.) at 37 °C.
Growth of isolate R1T on medium A containing 2 % of olive, sunflower, sesame, corn or rapeseed oil was determined by optical density measurements (OD600). All oils, except olive oil, which was from Sigma, were purchased from a local supermarket. Utilization of various other organic substrates as carbon and energy sources and other physiological features were determined by using the bioMérieux API 20A test kit according to the manufacturer's instructions. Growth rates, optimum growth temperature and tolerance to pH and NaCl were determined in medium A containing 1 % (w/v) yeast extract as the sole carbon and energy source. For these studies, cells were grown at temperatures ranging from 20 to 55 °C, at initial pH values of 4–9 (adjusted with 2·5 M NaOH or 2·5 M HCl) and in 0–6 % (w/v) NaCl for 7 days.
Cell morphology and size were examined using phase-contrast microscopy with a Nikon Optiphot-2 microscope at x1000 magnification. Gram staining was performed by using a Difco Gram stain set and cultures were grown in medium A according to standard procedures (Gerhardt et al., 1994). Spore formation was also determined by microscopy after staining with malachite green (Gerhardt et al., 1994). Mobility was determined under the phase-contrast microscope by making observations of samples from the liquid culture immediately after placing a cover slip over a drop of culture. Cells were also observed by a JSM-5600 LV scanning electron microscope (at x3000–13 000 magnification). For this purpose, cells were harvested from liquid culture during their exponential phase of growth, washed twice with water and dehydrated through a graded series of ethanol and isopropyl alcohol aqueous solutions. Cells were then mounted onto 12 mm cover slips, dried overnight in a vacuum desiccator and then gold-palladium (80/20) coated.
Genomic DNA was extracted and purified according to Arahal et al. (2002). Universal primers 28F (5'-AGAGTTTGATCCTGGCTCAG-3'; positions 8–28 using Escherichia coli numbering) and 1512R (5'-ACGGCTACCTTGTTACGACT-3'; positions 1512–1493 using E. coli numbering) were used to amplify the 16S rRNA gene (Weisburg et al., 1991). PCR products were purified by using the QIAquick PCR purification kit (Qiagen). DNA sequencing was performed on both strands by using the ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction kit (PE Biosystems) according to the manufacturer's instructions with an ABI Prism 3100 DNA analyser. GenBank and Ribosomal Database Project databases were used to search for 16S rRNA gene similarities (Maidak et al., 2000). Phylogenetic analysis based on the 16S rRNA gene sequence was performed with the aid of the DNAMAN 4.03 software package, using the neighbour-joining and Jukes–Cantor distance correction methods (Saitou & Nei, 1987). When constructing the phylogenetic tree, only sequences from the type strains of species whose names have been validly published were taken into account. The 16S rRNA gene sequence of C. tetanomorphum DSM 4474T was determined by the authors. An almost-complete 16S rRNA gene sequence (1439 bp; GenBank accession no. AY858804) of isolate R1T was used in the analysis.
DNA–DNA hybridization between isolate R1T and closely related type strains, as well as the determination of its DNA G+C content, was performed by the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ, Braunschweig, Germany).
Comparison of the partial 16S rRNA gene sequence of isolate R1T with those available in GenBank databases indicated that this isolate clustered with members of the genus Clostridium, cluster I (Fig. 1). The isolate had the highest identity with C. tetanomorphum (98 %) followed by Clostridium pascui (96 %) and Clostridium peptidivorans (94 %).
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Isolate R1T is mesophilic, exhibiting growth between 25 and 47 °C, with optimum growth at 37 °C. However, the growth rate at 45 °C was not significantly lower. The isolate grew optimally in the absence of NaCl but it tolerated up to 3 % (w/v) NaCl. The cells were able to grow in a pH range of 5·0–8·5 with a broad optimal pH for growth between 5·5 and 7·0. The growth temperature range of isolate R1T was similar to that of the type strain of C. tetanomorphum, but differed from that of the type strain of C. pascui, which grows between 10 and 43 °C. The optimum temperature was the same for all strains.
Isolate R1T also showed phenotypic differences when compared with C. tetanomorphum (Supplementary Table S1 available in IJSEM Online). Although lipase and indole production are common characteristics, utilization of glucose, xylose, maltose, sorbitol and salicin and hydrolysis of aesculin are not (Wilde et al., 1997). Regarding C. pascui, neither R1T nor those described as belonging to this species by Wilde et al. (1997) utilize glucose or other sugars, except for the type strain, which uses ribose weakly.
The DNA G+C content was 31·2 mol%, which differs from that of the type strains of C. tetanomorphum DSM 4474T and C. pascui DSM 10365T at 28 (Wilde et al., 1989) and 27 mol% (Wilde et al., 1997), respectively. The DNA–DNA relatedness was 61·9 and 54·3 % with C. pascui DSM 10365T and C. tetanomorphum DSM 4474T, respectively. DNA–DNA relatedness between the novel isolate and reference bacteria was significantly lower than the recommended value of 
70 %, which is accepted as the definition of distinct species (Wayne et al., 1987).
Based on the analysis of morphological, physiological and phylogenetic characteristics, isolate R1T should be classified in a novel species of the genus Clostridium, for which the name Clostridium lundense sp. nov. is proposed.
Description of Clostridium lundense sp. nov.
Clostridium lundense (lund.en'se. N.L. neut. adj. lundense from Lund, relating to the city where the type strain was isolated).
Cells are rod-shaped, 0·56x2·8–4·5 µm in size, Gram-positive and non-motile. Cells form associations of two or more cells in the stationary phase of growth. Colonies are circular, 1 mm in diameter, with entire margins and convex and have a cream colour when grown on peptone/yeast extract/glucose medium. Forms spores that are spherical, terminal and deform the cell shape. Obligately anaerobic, catalase-negative, indole-positive, glucosidase-positive, sulphide-production-positive. Gelatin is not hydrolysed but aesculin is. Growth occurs at temperatures between 25 and 47 °C, with optimum growth at 37 °C. Growth occurs in 0–3 % (w/v) NaCl but is optimal in the absence of NaCl. Growth occurs at pH 5·0–8·5 with a broad optimal pH for growth between 5·5 and 7·0. The strain does not utilize glucose, xylose, maltose, sorbitol, salicin, mannitol, lactose, sucrose, arabinose, glycerol, mannose, melezitose, raffinose, rhamnose or trehalose. The strain shows lipolytic activity; it hydrolyses olive, sesame and corn oils. The DNA G+C content is 31·2 mol%.
The type strain, R1T (=DSM 17049T=CCUG 50446T), was isolated from bovine rumen fluid.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Collins, M. D., Lawson, P. A., Willems, A., Cordoba, J. J., Fernandez-Garayzabal, J., Garcia, P., Cai, J., Hippe, H. & Farrow, J. A. E. (1994). The phylogeny of the genus Clostridium: proposal of five new genera and eleven new species combinations. Int J Syst Bacteriol 44, 812–826.
Dighe, A. S., Shouche, Y. S. & Ranade, D. R. (1998). Selenomonas lipolytica sp. nov., an obligately anaerobic bacterium possessing lipolytic activity. Int J Syst Bacteriol 48, 783–791.
Gerhardt, P., Murray, R. G. E., Wood, W. A. & Krieg, N. R. (editors) (1994). Methods for General and Molecular Bacteriology. Washington, DC: American Society for Microbiology.
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Jarvis, G. N., Strömpl, C., Moore, E. R. B. & Thiele, J. H. (1999). Isolation and characterisation of two glycerol-fermenting clostridial strains from a pilot scale anaerobic digester treating high lipid-content slaughterhouse waste. J Appl Microbiol 86, 412–420.[CrossRef][Medline]
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Markossian, S., Becker, P., Märkl, H. & Antranikian, G. (2000). Isolation and characterization of lipid-degrading Bacillus thermoleovorans IHI-91 from an Icelandic hot spring. Extremophiles 4, 365–371.[CrossRef][Medline]
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Svetlitshnyi, V., Rainey, F. & Wiegel, J. (1996). Thermosyntropha lipolytica gen. nov., sp. nov., a lipolytic, anaerobic, alkalitolerant, thermophilic bacterium utilizing short- and long-chain fatty acids in syntrophic coculture with a methanogenic archaeum. Int J Syst Bacteriol 46, 1131–1137.
Van Dyke, M. I. & McCarthy, A. J. (2002). Molecular biological detection and characterization of Clostridium populations in municipal landfill sites. Appl Environ Microbiol 68, 2049–2053.
van Gylswyk, N. O. & van der Toorn, J. J. T. K. (1987). Clostridium aerotolerans sp. nov., a xylanolytic bacterium from corn stover and from the rumina of sheep fed corn stover. Int J Syst Bacteriol 37, 102–105.
Wayne, L. G., Brenner, D. J., Colwell, R. R. & 9 other authors (1987). International Committee on Systematic Bacteriology. Report of the ad hoc committee on reconciliation of approaches to bacterial systematics. Int J Syst Bacteriol 37, 463–464.
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Wilde, E., Collins, M. D. & Hippe, H. (1997). Clostridium pascui sp. nov., a new glutamate-fermenting sporeformer from a pasture in Pakistan. Int J Syst Bacteriol 47, 164–170.
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