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VTT Biotechnology, PO Box 1500, Espoo, FI-02044 VTT, Finland
Correspondence
Riikka Juvonen
riikka.juvonen{at}vtt.fi
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93·9 % similarity). According to DNA–DNA hybridization results, the coccoid strains represented two genospecies, neither of which was related to any of the recognized Megasphaera species. Several phenotypic characteristics and/or DNA G+C content also differentiated the strains from each other and from their closest relatives. The other four novel strains were motile, slightly curved to helical rods, 0·6–0·8x3–50 µm or more in size. They shared identical 16S rRNA gene sequences and ribofragment patterns. The highest 16S rRNA gene similarity was found between these isolates and Pectinatus cerevisiiphilus ATCC 29359T (95·6 %) and Pectinatus frisingensis ATCC 33332T (93·6 %). The novel strains also differed from recognized Pectinatus species in their sugar utilization, proteolytic activity, catalase activity, antibiotic resistance and temperature tolerance. The results suggest that the bacteria belong to three novel species, for which the names Megasphaera paucivorans sp. nov. (type strain VTT E-032341T=DSM 16981T), Megasphaera sueciensis sp. nov. (type strain VTT E-97791T=DSM 17042T) and Pectinatus haikarae sp. nov. (type strain VTT E-88329T=DSM 16980T) are proposed.
Published online ahead of print on 9 December 2005 as DOI 10.1099/ijs.0.63699-0.
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strains VTT E-97791T, VTT E-032341T, VTT E-88329T and VTT E-90406T are DQ223729, DQ223730, DQ223731 and DQ217599, respectively.
Results of ribotyping analysis and volatile fatty acid profiles of the novel strains are available as supplementary material in IJSEM Online.
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). These Gram-negative, strictly anaerobic bacteria are affiliated to the Sporomusa sub-branch of the class ‘Clostridia’ in the phylum Firmicutes (Willems & Collins, 1995; Strömpl et al., 1999; Marchandin et al., 2003). At present, the genus Megasphaera Rogosa 1971 emend. Engelmann and Weiss 1985, emend. Marchandin et al. 2003 comprises three species: the type species Megasphaera elsdenii Rogosa 1971, first described as ‘Peptostreptococcus elsdenii’ (Gutierrez et al., 1959), Megasphaera cerevisiae Engelmann and Weiss 1986 and Megasphaera micronuciformis Marchandin et al. 2003. M. cerevisiae is the only brewery-associated species (Engelmann & Weiss, 1985; Suihko & Haikara, 2001). It spoils mainly low-alcohol beers by producing turbidity, H2S and short-chain fatty acids. At the time of writing, the genus Pectinatus Lee et al. 1978 comprises two brewery-contaminating species, Pectinatus cerevisiiphilus Lee et al. 1978 emend. Schleifer et al. 1990 and Pectinatus frisingensis Schleifer et al. 1990. They are common spoilage bacteria of unpasteurized packaged beers. Signs of spoilage include turbidity and off-flavours from the synthesis of organic acids and sulphuric compounds (Haikara & Helander, 2002). In an earlier study, we characterized 32 Megasphaera and Pectinatus strains of brewery origin, earlier identified by conventional methods, using ribotyping, specific PCR primers and partial 16S rRNA gene sequencing. The results indicated that four of the strains could represent a novel Pectinatus species and one strain could be a new member of the genus Megasphaera (Suihko & Haikara, 2001). Supporting our results, one of the Pectinatus strains, DSM 20764, has also been reported to exhibit low DNA relatedness (<20 % DNA–DNA reassociation value) with the previously described Pectinatus species and to contain cadaverine in its cell-wall peptidoglycan (http://www.dsmz.de/strains/no020764.htm), a typical feature for many members of the Sporomusa sub-branch (Schleifer et al., 1990; Strömpl et al., 1999). The aim of the present study was to investigate in further detail the taxonomic affiliation of the atypical brewery-contaminating strains, tentatively identified as Megasphaera and Pectinatus spp., and two novel coccoid-shaped isolates from spoiled beer.
Strains
Strains VTT E-032341T and VTT E-042576 originated from a spoiled Italian lager beer (pH 4·3) with an alcohol content of 5 % (v/v). Strain VTT E-97791T was isolated from a spoiled Swedish lager beer (pH 4·9) containing 2·8 % (v/v) alcohol. Strains VTT E-88329T and VTT E-88330 originated from air of a brewery bottling hall in Finland and VTT E-89371 from a spoiled Finnish lager beer containing 2·7 % (v/v) alcohol. The isolates were obtained from the samples by plating on peptone/yeast extract/fructose (PYF) medium (Suihko, 1999) followed by anaerobic incubation at 30 °C. Strain VTT E-97914 (=DSM 20764) was originally isolated from spoiled German beer and it was obtained from the DSMZ. The following reference strains were used: M. cerevisiae VTT E-79111T, M. elsdenii VTT E-84221T, M. micronuciformis VTT E-042113T (=AIP 412.00T, provided by H. Marchandin, CHU-Montpellier, France), P. frisingensis VTT E-79100T and P. cerevisiiphilus VTT E-79103T. The strains were subcultured on PYF or peptone/yeast extract/glucose (PYG) (for VTT E-97791T) medium (Suihko, 1999) at 30 °C, with the exception of VTT E-042113T, for which MRS agar (Oxoid) with 1 % fructose (BDH) and 25 % fermented wort without hops and an incubation temperature of 37 °C were used. Anaerobic conditions were created using a Whitley Anaerobic Cabinet MK III (Don Whitley Scientific) with 80 % N2, 10 % CO2 and 10 % H2 or Anoxomat WS8000 (Mart Microbiology) with 85 % N2, 5 % CO2 and 10 % H2.
Morphological characterization
Cell morphology (including motility) and Gram staining of 1-, 2- and 3-day-old broth cultures were examined by phase-contrast and normal light microscopy, respectively, at 800–1250x magnification (Polyvar; Reichert-Jung). Cell size ranges were expressed as mean width and length of 20 cells ± SD. Colony morphologies were determined after 3–4 and 7 days incubation on PYF or PYG plates. The results of the morphological characterization are presented in Table 1 and in the species descriptions below. The cellular characteristics of VTT E-97791T, VTT E-032341T and VTT E-042576 together with their isolation source and beer-spoilage ability suggested that they belong to the genus Megasphaera, which contains Gram-negative, non-motile, strictly anaerobic cocci (Rogosa, 1971; Engelmann & Weiss, 1985; Marchandin et al., 2003). Cells of the novel isolates were, however, smaller than those of M. elsdenii or M. cerevisiae and bigger than those of M. micronuciformis (Table 1). Strains VTT E-88329T, VTT E-88330, VTT E-89371 and VTT E-97914 resembled members of the genus Pectinatus morphologically (Schleifer et al., 1990). They formed an ‘X’ shape during movement, which is characteristic for P. frisingensis and P. cerevisiiphilus (Lee et al., 1978; Haikara et al., 1981) and discriminates them from morphologically similar Selenomonas species (Hespell et al., 1999).
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. The strains were ribotyped until the same pattern was generated three times. For 16S rRNA gene sequencing, DNA was isolated from 0·5–1 ml liquid cultures by bead-beating with 200 µm glass beads (0·1 g ml–1; Sigma) in a Ribolyser instrument (2 min at 6·5 m s–1; Hybaid) after washing of cells in ultra-pure water. Extracts (2–5 µl) were used directly as templates in 50 µl PCRs. The 16S rRNA gene was amplified according to Carlier et al. (2002) except that DyNAzyme II DNA polymerase (Finnzymes) was used. Resulting PCR products were purified using the QIAquick PCR Purification kit (Qiagen) and the manufacturer's procedure. Both strands of the gene were sequenced using the PCR primers and the primers described by Wilmotte et al. (1993) and ABI Prism BigDye Terminator version 3.0 or 3.1 Ready Reaction cycle sequencing kit (Applied Biosystems). Sequencing products were electrophoresed in the ABI Prism 310 Genetic Analyzer (Applied Biosystems). The sequences were compiled with the software DNAMAN version 4.1 (Lynnon BioSoft) and compared to GenBank/EMBL/DDBJ databases using BLAST (Altschul et al., 1997). The 16S rRNA gene sequences were aligned using Fast aligner. Phylogenetic relationships were inferred for representative strains (VTT E-97791T, VTT E-032341T and VTT E-88329T) using neighbour-joining (NJ; Saitou & Nei, 1987), maximum-parsimony (MP; Kluge & Farris, 1969) and maximum-likelihood (ML; Olsen et al., 1994) algorithms in the software package ARB (Strunk et al., 2000). Vertical gaps and ambiguous nucleotides were excluded from the analyses. Evolutionary distances for the NJ analysis were based on the correction of Jukes & Cantor (1969). Robustness of the NJ and MP tree topologies was estimated by bootstrapping with 1000 replications. DNA–DNA hybridization and G+C content analysis were done at DSMZ. Cells were disrupted with a French pressure cell (Thermo Spectronic) and purified on hydroxyapatite (Cashion et al., 1977). Hybridization was done according to De Ley et al. (1970) with the modifications of Huß et al. (1983) using a Cary 100 Bio UV/VIS spectrophotometer with in situ temperature probe (Varian). The DNA G+C content was determined by HPLC (adapted from Tamaoka & Komagata, 1984) of P1-nuclease-digested, dephosphorylated DNA (Mesbah et al., 1989) and calculated according to Mesbah et al. (1989). Genetic diversity of the novel isolates was studied by ribotyping in comparison with previously described Megasphaera and Pectinatus species. The results are available in Supplementary Figs S1 and S2 in IJSEM Online. The fingerprints distinguished VTT E-97791T from VTT E-032341T and VTT E-042576 (Supplementary Fig. S1). Strain VTT E-97791T grouped closely with M. cerevisiae, while strains VTT E-032341T and VTT E-042576 formed a distinct group with the highest similarity to the former strains and M. micronuciformis VTT E-042113T. The fingerprints of the rod-shaped strains (VTT E-88329T, VTT E-88330, VTT E-89371, VTT E-97914) grouped together and were clearly distinct from the fingerprints of recognized Pectinatus species (Supplementary Fig. S2). Hence, ribotyping results suggested that the coccoid and rod-shaped strains could respectively represent two and one novel species within the genera Megasphaera and Pectinatus.
Subsequently, nearly complete 16S rRNA gene sequences (1467–1556 nt) of the novel strains were determined except that the sequence of VTT E-97914 was taken from Sakamoto (1997). The 16S rRNA gene of Zymophilus raffinosivorans VTT E-90406T was also sequenced as it was not available from public databases. The sequences (1540 nt) of VTT E-032341T and VTT E-042576 shared 100 % similarity with each other and 99·3 % similarity with VTT E-97791T. Similarly, the sequences (1467 nt) of VTT E-88329T, VTT E-88330, VTT E-89371 and VTT E-97914 were identical. Based on BLAST searches, VTT E-032341T and VTT E-97791T shared the highest sequence similarity with M. micronuciformis (95·1 and 94·9 %, respectively), M. cerevisiae (94·6 and 94·5 %), M. elsdenii (93·2 and 93·3 %) and Anaeroglobus geminatus (93·2 and 93·3 %) over 1359 unambiguous bases. The sequences of the rod-shaped strains were most similar to those of P. cerevisiiphilus (96·3 %) and P. frisingensis (94·6 %). Thus, based on 16S rRNA gene sequence comparisons, all novel strains are members of the Sporomusa sub-branch of the class ‘Clostridia’, which contains low-G+C-content Gram-positive bacteria with Gram-negative cell walls (Willems & Collins, 1995; Strömpl et al., 1999).
In phylogenetic analyses with the most closely related species, VTT E-032341T and VTT E-97791T formed a well-supported group (100 % in the NJ and MP trees) that was linked to M. cerevisiae with good and moderate support, respectively, in the NJ (94 %; Fig. 1) and MP (67 %; not shown) trees. Based on the NJ analysis, the most closely related species to VTT E-032341T and VTT E-97791T were M. cerevisiae (93·9 and 93·9 %, respectively), M. micronuciformis (93·2 and 93·1 %), A. geminatus (90·6 and 90·5 %) and M. elsdenii (89·8 and 89·8 %). In accordance with Marchandin et al. (2003), the applied treeing methods could not resolve the exact branching order within the Megasphaera–Anaeroglobus group. The representative rod-shaped strain VTT E-88329T grouped with P. cerevisiiphilus in the NJ (97 %; Fig. 2), MP (57 %; not shown) and ML (not shown) analyses. These strains formed a robust cluster (100 % in the NJ and MP analyses) together with P. frisingensis and Pectinatus sp. B6. After the NJ analysis (Fig. 2), the sequence similarity of VTT E-88329T to P. cerevisiiphilus ATCC 29359T and P. frisingensis ATCC 33332T was 95·6 and 93·6 %, respectively. In conclusion, the phylogenetic analyses indicate that the new isolates represent novel species because sequence similarities to the closest relatives were below 97 %, the level below which strains are generally assigned to separate species (Stackebrandt & Goebel, 1994).
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). The results confirmed that VTT E-97791T and VTT E-032341T are not related to each other (41·0 %) or to M. micronuciformis VTT E-042113T (28·9 and 17·1 %, respectively), M. elsdenii VTT E-84221T (7·2 and 23·6 %) or M. cerevisiae VTT E-79111T (22·0 and 3·1 %) at the species level when the recommended criterion of 70 % DNA–DNA relatedness is applied for species delineation (Wayne et al., 1987; Stackebrandt & Goebel, 1994). The G+C content of the DNA of the proposed type strains of the novel species are shown in Table 1
or in the species descriptions below. The DNA G+C contents of VTT E-97791T and VTT E-88329T fall within the radiations observed for the genera Megasphaera and Pectinatus, respectively (Engelmann & Weiss, 1985; Schleifer et al., 1990; Marchandin et al., 2003). The DNA G+C content of VTT E-032341T is somewhat lower than reported for previously known Megasphaera species. The lower DNA G+C content clearly distinguishes both VTT E-97791T and VTT E-032341T from the closely related A. geminatus (Carlier et al., 2002).
Biochemical and physiological characterization
Biochemical and physiological characterization was performed using actively growing cultures, PYF medium and incubation at 30 °C, unless otherwise stated. All media were pre-reduced and all tests included positive and negative control strains. Nitrate reduction, arginine hydrolysis, urease activity, acetoin production and bile resistance were tested using diagnostic tablets and procedures from Rosco A/S. For antibiotic susceptibility tests, An-ident discs (Oxoid) were used. Gas production was determined in autoclaved PYF and PYG broth with inverted Durham tubes. Growth in MRS (Oxoid) and PY (Holdeman et al., 1977) media, in selective SMMP broth for Megasphaera and Pectinatus species (Lee, 1994) and in supplemented brucella blood agar (Jousimies-Somer et al., 2002) was also examined. In addition, the ability of selected strains (5x108 cells) to grow in different beers (330 ml, 0·3–4·6 % v/v alcohol, pH 4·1–4·5) was studied. Aerotolerance was examined by incubating plate cultures for 7 days in atmospheric air, in air with 10 % CO2 (Anoxomat WS8000) and under anaerobic conditions (control). For temperature limits of growth, plate cultures were incubated at 10, 15, 20, 30, 37, 40 or 45 °C and inspected after 7 and 14 days. Other growth tests were performed after Holdeman et al. (1977) or Jousimies-Somer et al. (2002). Short-chain fatty acids produced by the strains were extracted from 5-day-old broth cultures according to Jousimies-Somer et al. (2002). Volatile fatty acids were determined by GC as described by Suihko & Haikara (2001). Non-volatile acids were analysed by HPLC using the method of Rajakylä (1981) with minor modifications (oven temperature 35 °C, 5 mM sulphuric acid as a carrier liquid, flow rate 0·6 ml min–1).
Results of the physiological and biochemical tests are presented in Tables 1 and 2 and in the species descriptions below. None of the strains produced succinic, lactic or pyruvic acids. The volatile fatty acid profiles of the coccoid strains were dominated by C4–C6 acids (Supplementary Table S1 in IJSEM Online), which is characteristic for members of the genus Megasphaera (Engelmann & Weiss, 1985; Haikara & Lounatmaa, 1987; Jousimies-Somer et al., 2002; Marchandin et al., 2003), supporting their affiliation to this genus. However, strains VTT E-032341T and VTT E-97791 differed from the type strains of recognized Megasphaera species in that they produced isovaleric acid as the major end product. The fatty acid profiles of the type strains of M. cerevisiae and M. elsdenii determined in this study are in agreement with the results of Haikara & Lounatmaa (1987) and Suihko & Haikara (2001). The metabolic end products of M. micronuciformis VTT E-042113T were not analysed as it did not grow in PYG or PYF broth. No major differences were found in the volatile fatty acid profiles between the novel rod-shaped strains (VTT E-88329T, VTT E-88330, VTT E-89371, VTT E-97914) and the type strains of recognized Pectinatus species (Supplementary Table S2 in IJSEM Online). All strains also produced H2S and acetoin, but succinic, lactic and pyruvic acids were not detected. The production of acetate, propionate, H2S and acetoin as the major metabolites supports the affiliation of the novel strains to the genus Pectinatus and differentiates them from the closely related Zymophilus raffinosivorans and Zymophilus paucivorans, which do not produce acetoin, and Selenomonas lacticifex, which produces large quantities of lactate (Schleifer et al., 1990). The metabolic end products determined for the type strains of P. frisingensis and P. cerevisiiphilus in this study are in agreement with earlier published data (Lee et al., 1978; Haikara et al., 1981; Haikara, 1989; Schleifer et al., 1990).
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). The strains can also be differentiated from each other and from recognized Megasphaera species based on cell size, fermentation of carbohydrates, utilization of organic acids, gas production, antibiotic susceptibility, DNA G+C content (Table 1) and/or the sequence of the 16S rRNA gene. Furthermore, they can be discriminated from each other by ribotyping with EcoRI. The novel rod-shaped strains (VTT E-88329T, VTT E-88330, VTT E-89371, VTT E-97914) can be distinguished from the previously described Pectinatus species by ribotyping with EcoRI and by 16S rRNA gene sequencing. In addition, the following phenotypic characteristics differentiate them from P. frisingensis and P. cerevisiiphilus strains: inability to grow at 37 °C and ability to hydrolyse milk, positive catalase and negative urease reactions, vancomycin resistance and utilization of different organic acids and carbohydrates (Table 2). Based on these results, we propose two novel Megasphaera species, Megasphaera paucivorans sp. nov. and Megasphaera sueciensis sp. nov., and a novel Pectinatus species, Pectinatus haikarae sp. nov. As the original description of the genus Pectinatus by Lee et al. (1978) does not accommodate all of the phenotypic properties of P. haikarae, an emended genus description is proposed.
Description of Megasphaera paucivorans sp. nov.
Megasphaera paucivorans (pau.ci.vo'rans. L. adj. paucus few, little, L. part. adj. vorans devouring; N.L. part. adj. paucivorans devouring few substrates).
Gram-negative, non-spore-forming and non-motile cocci with a mean size of 1·5x1·2 µm, mainly arranged in pairs. Stationary phase cells may form chains of 20–25 diplococci and cell clumps. Strictly anaerobic. Moderate to good growth (2+ or 3+ on a scale of 0 to 4+) is obtained in autoclaved PYF, PYG, MRS and SMMP media. Growth in SMMP is accompanied by a colour change from violet to yellow. Poor growth in PY medium (1+), but the addition of 1 % (w/v) pyruvate or gluconate results in heavily turbid suspension (4+). Colonies on PYF plates appear after 3 days at 30 °C and after 7 days are yellowish, circular, convex, glossy and opaque with entire margins and a diameter of 1–1·5 mm. Grows at 15–37 °C, with an optimum at around 30 °C, but not at 10 or 45 °C. Major volatile fatty acids produced in beer are butyric and isovaleric acids; H2S and minor amounts of propionic, isobutyric, valeric and caproic acids are also produced. Other phenotypic properties and characteristics that differentiate the species from other Megasphaera species are listed in Table 1.
The type strain, VTT E-032341T (=DSM 16981T), was isolated from spoiled Italian beer.
Description of Megasphaera sueciensis sp. nov.
Megasphaera sueciensis (sue.ci.en'sis. N.L. fem. adj. sueciensis pertaining to Sweden).
Strictly anaerobic, Gram-negative, non-spore-forming and non-motile cocci, mainly arranged in pairs and occasionally in short chains. Mean cell size is 1·2x1·0 µm. Moderate growth (2+ on a scale of 0 to 4+) is obtained in autoclaved PYF, PYG, MRS and SMMP media at 30 °C. The addition of 1 % (w/v) pyruvate or gluconate to the PY medium markedly stimulates growth. Colonies on PYF and PYG plates appear after 4 days at 30 °C and after 7 days are slightly yellowish, glossy, convex, opaque, smooth and circular with entire edges and a diameter of 0·5–0·8 mm. Grows at 15–37 °C, with an optimum at around 30 °C, but not at 10 or 45 °C. Other physiological properties and characteristics that differentiate this species from other Megasphaera species are shown in Table 1.
The type strain, VTT E-97791T (=DSM 17042T), was isolated from spoiled Swedish beer.
Emended description of Pectinatus Lee et al. 1978
Pectinatus [Pec.ti.na'tus. L. part. adj. pectinatus combed (bacteria)].
Cells are non-spore-forming, slightly curved to helical rods, 0·4–0·9x2–50 µm or more, with rounded ends and a Gram-negative cell wall. They occur singly, in pairs or rarely in short chains. Cells are usually motile by means of comb-like flagellation which emanates from only one side of a cell. Cadaverine or putrescine is found in the cell-wall peptidoglycan. Organisms are strictly anaerobic mesophiles with fermentative metabolism. Glucose and fructose are mainly metabolized to acetic and propionic acids. H2S and acetoin and occasionally minor amounts of succinic acid are produced. Cells do not synthesize cytochrome oxidase, desulfoviridin or indole, hydrolyse arginine or gelatin or reduce nitrate. The G+C content of the DNA is 38–41 mol%. The species of the genus can be separated from each other by using various genetic and phenotypic criteria. Isolated from beer and brewing processes. The type species is Pectinatus cerevisiiphilus Lee et al. 1978 emend. Schleifer et al. 1990.
Description of Pectinatus haikarae sp. nov.
Pectinatus haikarae (hai.ka'rae. N.L. gen. fem. n. haikarae of Haikara, named after Dr Auli Haikara for her many contributions to the characterization and detection of Pectinatus species).
Cells are Gram-negative, non-spore-forming, straight or slightly curved, flexible rods with rounded ends, 0·6–0·8x3–50 µm or more in size. They usually occur singly and occasionally in pairs. Stationary-phase cells may form long, helical filaments and round and loop-shaped formations or have club-shaped, distended ends. Young cells are motile, following an X-like pattern. Old cells exhibit slow, snake-like movements or are non-motile. Strictly anaerobic. Grows at 15–30 °C, but not at 10 or 37 °C. The optimum lies between 20 and 30 °C. Good growth (3+ or 4+ on a scale of 0 to 4+) is obtained in PYF, PYG, MRS and SMMP media after 1–2 days at 30 °C. Weak growth on supplemented brucella blood agar and PY medium. Colonies on PYF and PYG plates after 3 days at 30 °C are convex to pyramidal, glossy, opaque, cream to grey in colour and circular with entire margins and a diameter of 0·5–2·5 mm. Major products of fructose fermentation are propionic and acetic acids. Acetoin and H2S are also produced. Differential characteristics compared with other Pectinatus species are shown in Table 2.
The type strain is VTT E-88329T (=DSM 16980T), with the DNA G+C content of 38·8 mol% (Tm), isolated from a brewery bottling hall in Finland. Strains have also been isolated from spoiled German and Finnish beers.
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ACKNOWLEDGEMENTS |
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-D-melibiose, xylitol, succinate and pyruvate. Data for AIP 313.00T are from Carlier etal. (2002)
, L-arabinoseabc, DL-erythritolabcde, D-fructoseabcde, D-galactoseabc, D-glucoseabcde, glycerolabcde, mannoseb, rhamnoseabcde, D-riboseabcde and utilization of DL-lactatebce. All strains were negative (except those in parentheses) for acid production from glycogenabcde, inulinabce, maltoseac (VTT E-97914, VTT E-79100T), melezitoseabce and raffinoseabcde, utilization of succinate