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Soehngenia Saccharolytica Gen. Nov., Sp. Nov. and Clostridium Amygdalinum Sp

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Soehngenia Saccharolytica Gen. Nov., Sp. Nov. and Clostridium Amygdalinum Sp View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Wageningen University & Research Publications International Journal of Systematic and Evolutionary Microbiology (2003), 53, 1791–1799 DOI 10.1099/ijs.0.02668-0 Soehngenia saccharolytica gen. nov., sp. nov. and Clostridium amygdalinum sp. nov., two novel anaerobic, benzaldehyde-converting bacteria Sofia N. Parshina,1,2,3 Robbert Kleerebezem,2 Jose Luis Sanz,3 Gatze Lettinga,2 Alla N. Nozhevnikova,1 Nadezhda A. Kostrikina,1 Anatoly M. Lysenko1 and Alfons J. M. Stams3 Correspondence 1Laboratory of Microbiology of Anthropogenic Environments, Institute of Microbiology, Russian Sofia N. Parshina Academy of Sciences, Moscow, Russia [email protected] 2,3Subdepartment of Environmental Technology2 and Laboratory of Microbiology3, Wageningen University, Wageningen, The Netherlands Two anaerobic, benzaldehyde-converting bacteria were isolated from an anaerobic upflow anaerobic sludge bed (UASB)-reactor treating potato starch waste water. Strain BOR-YT converted benzaldehyde to benzoate and benzylalcohol in approximately equimolar concentrations. Benzaldehyde conversion did not support growth. Strain BOR-YT was Gram-positive and rod-shaped, and its cells were slightly thickened in the middle. The strain was a mesophilic spore-former that grew between 15 and 40 6C, with optimum growth at 30–37 6C. The optimum pH for growth was pH 7?0. Strain BOR-YT grew on a wide range of carbohydrates and some other carbon sources including yeast extract, cysteine and serine. The G+C content of its DNA was 42 mol%. According to physiological characteristics and 16S rRNA gene sequence analysis, confirmed by DNA–DNA hybridization with its phylogenetic neighbours, strain BOR-YT belongs to a novel genus of cluster XII of the clostridia, namely Soehngenia; the name Soehngenia saccharolytica is proposed for the type species (type strain BOR-YT=DSM 12858T=ATCC BAA-502T). Strain BR-10T reduced benzaldehyde to benzylalcohol. This conversion was coupled to growth. In a medium containing yeast extract, the presence of benzaldehyde resulted in the accumulation of more than twofold more cells. Strain BR-10T was a Gram-positive organism that was characterized by oval- or rod-shaped cells with oval ends, which occurred singly, in pairs or sometimes in chains. The strain was moderately thermophilic and grew between 20 and 60 6C, with optimum growth at 45 6C. The optimum pH for growth was between pH 7?0 and 7?5. Strain BR-10T grew on a wide range of carbon sources including carbohydrates, yeast extract, casein and some amino acids. The G+C content of its DNA was 32 mol%. As determined by 16S rRNA gene sequence analysis, strain BR-10T represents a novel species of cluster XIVa of the clostridia; the name Clostridium amygdalinum is proposed for this novel species (type strain BR-10T=DSM 12857T=ATCC BAA-501T). INTRODUCTION the aldehyde group of vanillin to the carboxyl level (Lux et al., 1990). Ruminococcus productus (formerly Peptostrep- Many anaerobic bacterial strains are able to convert tococcus productus) reduced the aldehyde group of vanillin aromatic aldehydes to other compounds (Krumholz & when co-cultured with CO but was not capable of this reac- Bryant, 1985; Lux et al., 1990; Sembring & Winter, 1990; tion when vanillin was the sole substrate (Lux et al., 1990). Lux & Drake, 1992; Go¨ßner et al., 1994). Moorella thermo- acetica (formerly Clostridium thermaceticum) (Collins et al., Clostridium aceticum oxidized aromatic aldehydes (Lux 1994) and Clostridium formicaceticum were shown to oxidize & Drake, 1992). A few Desulfovibrio strains oxidized the aldehyde group of vanillin and other aromatic aldehydes The GenBank/EMBL/DDBJ accession numbers for the 16S rDNA with sulfate as an electron acceptor (Zellner et al., 1990). sequences of Soehngenia saccharolytica BOR-YT and Clostridium A limited number of the strains were tested for their amygdalinum BR-10T are AY353956 and AY353957, respectively. ability to convert benzaldehyde. Clostridium acetobutylicum Electron micrographs of Soehngenia saccharolytica BOR-YT (Fig. I) and reduced benzaldehyde, but 10 mM benzaldehyde caused Clostridium amygdalinum BR-10T (Fig. II) are available in IJSEM Online. inhibition of growth. This bacterium required the presence 02668 G 2003 IUMS Printed in Great Britain 1791 S. N. Parshina and others of glucose and butyrate in the medium (Green et al., 1994). et al., 1991). Gram staining was done according to standard procedures Desulfovibrio desulfuricans both oxidized and reduced (Doetsch, 1981). benzaldehyde in the presence of nitrate (Parekh et al., 1996). Analytical methods. Aromatic compounds (benzaldehyde, benzo- ate and benzylalcohol) and volatile fatty acids were analysed as In a previous study (Parshina et al., 2000), the isolation of described previously (Parshina et al., 2000). Organic acids were two bacterial strains able to convert benzaldehyde in the analysed by HPLC (Merck) with an RI-detector. A Polyspher OAHY absence of inorganic electron acceptors was described. The column (300 by 6?5 mm) was used. Hydrogen and carbon dioxide bacteria were isolated from an anaerobic upflow anaerobic were analysed by using a Chrompack gas chromatograph (CP9001) sludge bed (UASB)-reactor treating potato starch waste equipped with a TCD-detector. The stainless steel column was filled water. One bacterium (strain BOR-YT) performs the with Molsieve 13X (60–80 mesh). The TCD-detector run was at 100 uC; argon was used as the carrier gas. Sulfide was analysed by dismutation of benzaldehyde to benzoate and benzylal- the method Tru¨per & Schlegel (1964). cohol. The other bacterium (strain BR-10T) uses benzalde- hyde as an electron acceptor resulting in the formation of Determination of temperature and pH optima. For the deter- mination of temperature optima, strain BOR-YT was cultivated in a benzylalcohol and obtains metabolic energy from this 21 21 21 medium containing 2 g yeast extract l plus 2 g glucose l , and reaction. In a medium containing 1 g yeast extract l , strain BR-10T was cultivated in a medium containing 2 g yeast the addition of 10 mM benzaldehyde resulted in a twofold extract l21 plus 10 mmol crotonate l21. Strain BOR-YT was incu- T higher number of strain BR-10 cells. The mechanisms of bated at 5–52 uC and strain BR-10T was incubated at 10–70 uC. pH benzaldehyde conversion by strains BOR-YT and BR-10T optima were determined by incubating strains BOR-YT and BR-10T have not been described. Therefore, it was expected that at 30 and 45 uC, respectively, at initial pH values ranging from the two strains represented novel micro-organisms. pH 5?0to9?0. The pH was adjusted by adding 6 M HCl or 6 M NaOH. In this report, we describe the phenotypic and phylogenetic Physiological tests. The following substrates were tested as characteristics of the mesophilic bacterium which converts carbon and energy sources (20 mmol l21 each, unless indicated): benzaldehyde to benzoate and benzylalcohol (strain BOR- yeast extract (2 g l21), formate, acetate, propionate, isobutyrate, YT) and of the moderately thermophilic bacterium able glucose, fructose, sucrose, xylose, arabinose, rhamnose, mannose, to reduce benzaldehyde to benzylalcohol (strain BR-10T). ribose, maltose, cellobiose, galactose, melibiose, lactose, cellulose, On the basis of their phenotypic and phylogenetic charac- xylan, mannitol, casitone, inositol, methanol, ethanol, ethylene glycol, ethylamine, lactate, succinate, fumarate, crotonate, pyruvate, teristics, it is proposed that these two bacteria be named malate, starch (2 g l21), glycerol, cysteine, serine, arginine, leucine, Soehngenia saccharolytica and Clostridium amygdalinum, glycine, alanine, glutamate, methionine, casein (2 g l21), casein 21 21 respectively. hydrolysate (2 g l ), peptone (2 g l ), gelatin, betaine, H2/CO2 [80 % : 20 % (v/v) in the gas phase], creatine and creatinine. To investigate the utilization of electron acceptors, strains were culti- METHODS vated in a medium containing 2 g yeast extract l21. The following electron acceptors were tested: Na2SO4 (10 mM), Na2SO3 (2 mM), Organisms and isolation of the strains. An enrichment culture 0 Na2S2O3 (10 mM), Na2S2O4 (10 mM), Na2S2O5 (10 mM), S was obtained from a mesophilic laboratory-scale UASB-reactor treat- (2 g l21) and NaNO (2 g l21). To test the ability of molecular ing potato starch waste water. The isolation of strains BOR-YT and 3 T nitrogen fixation, the medium described by Skinner (1971) was BR-10 from this enrichment has been described previously 21 T T used. Standard medium supplemented with 2 g yeast extract l (Parshina et al., 2000). Clostridium ultunense BS (DSM 10521 ) was plus 10 mM pyruvate flushed with N served as a control. The ¨ 2 kindly provided by Dr Anna Schnurer (Department of Micro- aerotolerance of the strains was determined as described for biology, Swedish University of Agricultural Sciences, P.O. Box 7025, T Clostridium aerotolerans (van Gylswyk & van der Toorn, 1987) in SE-750 07 Uppsala, Sweden); Tissierella creatinini DSM 9508 was the medium supplied with 20 mM glucose. The air was injected into obtained from the DSMZ (Deutsche Sammlung von Mikroorganismen the closed bottles through a membrane filter. The volume of the und Zellkulturen, Braunschweig, Germany). air was 0?5–100 % (v/v). Media and cultivation. Different media were used for cultivation Biochemical tests. Several physiological and biochemical charac- of the isolates and the culture collection strains. A bicarbonate- teristics of the cultures were analysed using API 20 E biochemical phosphate-buffered medium was used. Medium preparation and kits (Identification system for Enterobacteriaceae and other Gram- cultivation of the isolates has been described previously (Parshina negative rods; bioMe´rieux). To test for the presence of catalase, cell et al., 2000). Strain BOR-YT was grown at 30 uC and strain BR-10T material was exposed to 10 % H2O2. was grown at 45 uC. The bacteria were cultivated routinely in 120 ml serum bottles containing 50 ml medium. The amount of inoculum Isolation of genomic DNA. Wet biomass was washed with a solu- used was 1–2 %.
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