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Lactobacillus graminis sp. nov., a New Species of Facultatively Heterofermentative Lactobacilli Surviving at Low pH in Grass Silage
System. Appi. Microbioi. 10, 279-283 (1988)
Lactobacillus graminis sp. nov., a New Species of Facultatively Heterofermentative Lactobacilli Surviving at Low pH in Grass Silage R. BECK!, N. WEISS 2, and]. WINTER3 1
2
3
Bayerische Landesanstalt rur Bodenkultur und Pflanzenbau, D-8000 Munchen 19, Federal Republic of Germany Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, D-3300 Braunschweig, Federal Republic of Germany Institut rur Mikrobiologie, Universitiit Regensburg, D-8400 Regensburg, Federal Republic of Germany Received February 10, 1988
Summary A group of 11 facultatively heterofermentative rod-shaped bacteria were isolated at a late fermentation stage of pre-wilted grass. These bacteria were found to differ from currently described lactobacilli in the spectrum of fermented sugars, in particular in their ability to ferment xylose. The isolates were similar to Lactobacillus curvatus and Lactobacillus sake with respect to D( -)- and L( +)- lactate formation, Rr values of the D- and L-LDH, G+C content of the DNA and the L-Lys-D-Asp murein type. Teichoic acid was lacking. DNA-DNA homology among the isolates was high, while low homology values were obtained with the type strains of L. curvatus and L. sake, indicating only a distant relationship. It is proposed that the isolates represent a new species named Lactobacillus graminis sp. nov.
Key words: Lactobacillus graminis sp. nov. - Lactobacilli - Taxonomy - DNA- DNA-homology - Grass silage - Population
Introduction The microbial ecology of fermenting vegetable material, including silages, and the use of bacterial inoculants as silage additives (starter cultures) has been reviewed recently (Seale, 1986; Daeschel et ai. 1987). The development of the flora of lactic acid bacteria during ensilage of different crops was found to be influenced mainly by the dry matter content of the crops and by the time of fermentation (Wieringa, 1960). The succession of single species has been studied during ensilage of wheat (Moon et aI., 1981) and, more recently, of ammonia-treated straw (Suhaimi et aI., 1987a). Straw fermentation required the enrichment of an alkaline-adapted association of streptococci and lactobacilli (Suhaimi et aI., 1987b). In earlier studies on the taxonomy of facultatively heterofermentative lactobacilli (as defined by Schleifer, 1987) from silage, few differences were noted between the isolates (Abo-Elnaga and Kandler, 1965a, b; Beck, 1965; Beck et aI., 1987a, b; Bucher, 1970). In this contribution we report the results of our studies on the physiology of the ensilage process of grass and the succession of lactobacilli during this process. A large number of lactobacilli
was isolated. One group of these from a late phase of the grass fermentation differed from currently described species and thus was studied in detail. These isolates are considered to belong to a new species of lactobacilli, for which the name Lactobacillus graminis sp. nov. is proposed. Material and Methods Organisms. The type strains of Lactobacillus curvatus (DSM 20019) and Lactobacillus sake (DSM 20017) were obtained from the Deutsche Sammlung von Mikroorganismen, D-3300 Braunschweig-Stockheim. Medium and growth conditions. Isolates from grass silage were obtained by repeated plating of diluted samples of silage juice on MRS-agar (DeMan et ai., 1960). Type strains and all isolates were cultured in MRS-medium (DeMan et ai., 1960) at 28°C. Stock cultures were kept in conservation agar stabs (Wieringa, 1960) at 4°C. Determination of the spectrum of fermented substrates. The ability of the isolates to ferment different sugars was tested ac-
280
R. Beck, N. Weiss, and J. Winter
cording to Rogosa and Sharpe (1959), Sharpe (1962) and Rogosa (1970). Sugar solutions were filter-sterilized and added to the sugar-free basal medium to give a final concentration of 1 gil. Disc electrophoresis.The electrophoretic mobility of the lactate dehydrogenase (LDH) was determined in polyacrylamide gels according to Maurer (1968), as modified by Stetter and Kandler (1973). Rabbit L-lactate dehydrogenase isoI served as a reference. Analysis of fermentation products. The configuration and the amount of lactic acid was determined enzymatically according to Horost (1970), using a test kit with either L- or D-lactate dehydrogenase. Acetic acid was determined with acetate kinase according to Ho/z and Bergmeyer (1970). The amount of ethanol was quantified using ethanol dehydrogenase and acetaldehyde dehydrogenase as described by Beutler and Michal (1977). Cell wall analysis. Cell walls were prepared and the murein components determined according to Schleifer and Kandler (1967, 1972). Teichoic acids were extracted from cell walls using the hydrofluoric acid (HF) method described by Fiedler et al. (1981). Sugar alcohols were separated as the corresponding alditol-acetate-derivatives (Albersheimer et al., 1967) by gas-liquid chromatography. Determination of the G+C content of the DNA and DNADNA homology studies. DNA was extracted and purified according to a modified method of Marmur (1961). The melting point (T M) of the DNA was determined according to Marmur and Doty (1962) and the G+C mol% was calculated using the equation of DeLey (1970). DNA-DNA hybridization experiments and the calculation of the % homology values were carried out as described by DeLey et al. (1970), Gillis et al. (1970) and Huss et al. (1983).
Results and Discussion Succession of lactobacilli and acid formation during the ensilage process of pre-wilted grass
Pre-wilted meadow grass with a dry matter content of 47.6% was chopped and ensilaged at 25°e in several laboratory silos of 11 capacity for 90 days. Two silos each were opened for chemical and microbiological analyses at different intervals. Lactic acid production reached the maximum after 30 days, while the maximum acetic acid concentration was reached after only 1 day (Fig. 1). The majority of the grass protein was degraded within one week, as judged from'ammonia production. Protein degradation was slow, but almost linearly proceeding for three months, while the maximum amount of lactic acid was reached after 1 month (Fig. 1). The number of total viable colony forming units (cfu,) was highest after 7 days (108/g) and declined to 107/g after 30 days and less than 106/g after 90 days (Fig. 1). To determine the succession of different species or groups of lactic acid bacteria (viable cells) during the ensilage process, samples were serially diluted and streaked on MRS-agar plates. After incubation at 28°e, 10 colonies of each distinguishable colony type from plates containing approximately 100 colonies, were inoculated into liquid medium. A rough classification of the isolates was performed by testing for the following features: morphology, colour of colonies, gas formation (with Durham tubes), growth at 15 and 45 °e, formation of D( -)-, L( +)- or DILlactate, NH3 production from arginine and by the spectrum of fermented sugars. Furthermore, the presence/abs-
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Fig. 1. Kinetics of acetate-, D/L-lactate and ammonia formation and total number of viable population during ensilage of prewilted grass.
ence of m-DAP in the cell walls was analysed. The succession of Streptococcaceae and lactobacilli, tentatively identified according to Teuber and Geis (1981) and Sharpe (1981) is shown in Fig. 2. Initially streptococci (= lactococci; Schleifer, 1987), Lactobacillus coprophilus, Pediococcus pentosaceus and Leuconostoc sp. were predominant. Only when these organisms had lost viability, were other strains detected which could not be classified as strains of a known species of homofermentative lactobacilli, mainly due to their pentose utilization pattern. Upon further characterization these strains turned out as members of a new species, named Lactobacillus gram in is spec. nov. L. graminis strains may have been present in earlier stages of fermentation, but not in sufficiently high numbers to be detected. L. graminis comprised approximately 10% of the viable population after 30 days but up to 40% after 90 days. Together with Leuconostoc sp., Lactobacillus plantarum and P. pentosaceus, it appeared to survive longer in the silage than other lactobacilli. Eleven strains of L. graminis were isolated from samples drawn at day 30 and day 90. Five of these strains were further characterized.
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Lactobacillus graminis sp. nov.
Characteristics of Lactobacillus graminis sp. nov.
All isolates, identified as L. graminis formed smooth, non-pigmented round colonies of 1-2 mm in diameter. In liquid cultures the rod-shaped, slightly curved cells, 0.7-1 by 1.5-2 [.Lm in size, occured singly, in pairs or in chains up to 10 [.Lm (Fig. 3) A flocculant sediment was formed after 3 days of growth. Cells stained Gram-positive. A comparison of physiological features of Lactobacillus graminis isolates with those of other lactobacilli is shown in Table 1. The L-Lys-D-Asp murein type (4 isolates tested), the G+C content of the DNA (5 isolates tested, 41-43 mol% G+C) and the electrophoretic mobility of the lactate dehydrogenases of 5 isolates of L. graminis were identical. On the basis of these properties L. graminis isolates could not be differentiated from L. curvatus and L. sake. However, strains of L. graminis differed from L. curvatus and L. sake in several other characteristics, e. g. the ability to ferment maltose, ribose and xylose (Table 1). The inability to ferment xylose is considered to be an important constant feature of the facultatively heterofermentative lactobacilli (Schleifer, 1987). Fermentation balances
Glucose and xylose fermentation by two of the eleven isolates of L. graminis was analysed. One mol of glucose was almost stoichiometrically fermented to 2 mol D/Llactate. Only a small amount of acetate was formed (Table 2). One mol xylose was fermented to 1 mol D/L-Iactate and 1 mol acetate. No ethanol was produced. A similar fermentation balance was found for arabinose and ribose fermentation by L. plantarum (Table 2). In very young cultures (growth time 6 h) of both L. graminis strains tested, L-Iactate predominated over (only traces of) Dlactate, but an almost racemic mixture was found in stationary cultures.
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281
282
R. Beck, N. Weiss, and J. Winter
Species
Substrate
L-Lactate
V
Table 2. Fermentation balance of glucose, xylose, ribose and arabinose by resting cells of L. graminis and L. plantarum
Acetate
a
b
a
b
a
b
31,1 11,1
0,89 0,36
3,3 29,6
0,095 0,95
0,4 0,15
0,4 18,4
0,065 0,98
0,71 0,37 0,33
0,2 28,8 21,3
0,007 0,72 0,665
L. graminis G30 (5)
Glucose Xylose
35 31
37,5 21,4
1.07 0,69
L. graminis G90 (1)
Glucose Xylose
6,5 18,7
10,1 12,8
1,55 0,68
L. plantarum
Glucose Arabinose Ribose
28,3 40,0 32,0
43,5 26,4 21,4
1,54 0,66 0,67
R30 (19)
D-Lactate
2.6 2,85 20,2 14,7 10,6
V = Fermented substrate in mmolJl a = produced acid in mmolll (total) b = produced acid in mmolJl per fermented mmolJl substrate
Genomic characteristics L. graminis strains
Gram-positive, non-motile, slightly curved rods with
The G+C content of the DNA of L. graminis strain G 90(1) was 42 mol%. The G+C content for 4 other L. graminis strains varied between 41-43 mol%. DNA-DNA hybridisation studies showed a high homology (90-100%) among the five strains of L. graminis (Table 3) tested, but low values between the L. graminis strains and the type strains of L. curvatus and L. sake (20% and 40%, respectively). These low hybridisation values indicate only a distant relationship between L. gram in is, L. curvatus and L. sake and, together with differences in the spectrum of fermented sugars, define the new species L. graminis. Description of Lactobacillus graminis sp. nov. Lactobacillus graminis sp. nov., gra'mi.nis. L. n. gramen, grass, graminis of grass.
r~)Unde? en?s, g~nerally 0.7:-1 by 1.5-2 f.lm, occuring
smgly, m paIrS or m short chams, slightly curved. Colonies smooth, round, non-pigmented. Flocculant sediment after three days of growth in MRS-broth. Facultatively heterofermentative. Produce exclusively DIL-Iactate from glucose and D/L-Iactate and acetate from xylose. Mic.roaerophilic. No growth at 45°C, growth at 15 0c. Opnmal temperature 30-35°C. Fermentation of carbohydrates and other characteristics are as given in Table 1. The peptidoglycan is of the L-Lys-D-Asp type. Cell wall teichoic acid not detectable. The G+ C content of the DNA is in the range 41-43 mol%, for strain G90(1) 42 mol% (TM ). Habitat: grass silage. Type strain: Lactobacillus graminis strain G90(1) DSM 20719. '
Table 3. DNA-DNA-hybridisation (% homology) of L. graminis strains and related lactobacilli
L. sake 27/85
L. graminis strains G30(13) G90(8)
L. sake
L. curvatus
20017
20019
G90(1)
78
41.5
27.3
40
39.4
32.8
30.6
28
36
37.2
48
35
G30(5)
G90(3)
L. sake
27/85
100
L. sake
20017
100
L. curvatus
20019
100
L. graminis
G90(1) G30(5) G90(8) ".. )lot -4etermined
100
100 100
94.5 100 100
94.8 96.4 91.5
97.2
Lactobacillus graminis sp. nov.
References Abo-Elnaga, ]. G., Kandler, 0.: Zur Taxonomie der Gattung Lactobacillus Beijerinck. I: Das Subgenus Streptobacterium Orla-Jensen. Zbl. Bakt., II Abt. 119, 1-36 (1965a) Abo-Elnaga, ]. G., Kandler, 0.: Zur Taxonomie der Gattung Lactobacillus Beijerinck. II: Das Subgenus Betabacterium Orla-Jensen. Zbl. Bakt., II Abt. 119, 177-129 (1965b) Albersheim, P., Nevois, D.]., Englis, P. D., Karr, A.: A method for the analysis of sugars in plant cell wall polysaccharides by gas-liquid chromatography. Carbohydr. Res. 5, 340-345 (1967) Beck, Th.: Untersuchungen iiber die Okologie und Physiologie der Giirfuttermikroflora. Landw. Forschung 19, 3-11 (1965) Beck, R., Grop, F., Beck, Th.: Untersuchungen zur Kenntnis der Gatfuttermikroflora, I. Mitteilung: Die Zusammensetzung der Milchsaurebakterienpopulation bei der Vergarung von Deutschem Weidelgras und Rotklee. Z. Das wirtschaftseigene Futter 33, 13-33 (1987a) Beck, Th., Grop, F., Beck, R.: Untersuchungen zur Kenntnis der Garfuttermikroflora, II. Mitteilung: Milchsaurebakterien von Silagen unterschiedlicher Qualitat aus Praxissilagen. Z. Das wirtschaftseigene Futter 33, 34-43 (1987b) Beutler, H. 0., Michal, G.: Neue Methode zur enzymatischen Bestimmung von Athanol in Lebensmitteln. Fresenius J. Ana!. Chern. 284,113-117 (1977) Bucher, E.: Beitriige zur Mikrobiologie der Silagegarung und der Garfutterstabilitat. Dissertation, Universitat Miinchen 1970 Daeschel, M. A., Anderson, R. E., Fleming, H. P.: Microbial ecology of fermenting plant materia!' FEMS Microbio!. Rev. 46, 357-367 (1987) DeLey, ].: Reexamination oJ the association between melting point, buoyant density, and chemical base composition of deoxyribonucleic acid. J. Bact. 101, 738-754 (1970) DeMan, ]. C., Rogosa, M., Sharpe, M. E.: A medium for the cultivation of lactobacilli. J. App!. Bact. 23, 130-135 (1960) Fiedler, F., Schaffler, M. ]., Stackebrandt, E.: Biochemical and nucleic acid hybridisation studies on Brevibacterium linens and related strains. Arch. Microbiol. 129, 85-91 (1981) Gillis, M., De Ley, ]., DeCleene, M.: The determination of molecular weight of bacterial genome DNA from renaturation rates. Europ. J. Biochem. 12, 143-153 (1970) Holz, G., Bergmeyer, H. U.: Acetatbestimmung. In: Methoden der enzymatischen Analyse (H. U. Bermeyer, ed.). Weinheim, Verlag Chemie 1970 Horost, H. ].: L-Lactatbestimmung. In: Methoden der enzymatischen Analyse (H. U. Bergmeyer, ed.), pp.266-270. Weinheim, Verlag Chemie 1970 Huss, V. A . R., Fest, R., Schleifer, K. H.: Studies on the spectrophotometric determination of DNA hybridization from renaturation rates. System. Appl. Microbiol. 4, 184-192 (1983) Kandler, 0., Weiss, N.: Genus Lactobacillus. In: Bergey's Manual of Systematic Bacteriology, Vol. II (P. H. A. Sneath and]. G.
283
Holt, eds.), p. 1208. Baltimore, The Williams and Wilkens Co. 1986 Marmur, ].: A procedure for the isolation of DNA from microorganisms. J. Molec. BioI. 3, 208-218 (1961) Marmur, ]., Doty, P.: Determination of the base composition of deoxyribonucleic acid from its thermal denaturation temperature. J. Molec. Bio!. 5, 109-118 (1962) Maurer, H. R.: Disk-Elektrophorese. Berlin, Walter de Gruyter Verlag 1968 Moon, N. ]., Moon, L. c., Ely, L. 0., Parker, ]. A.: Lactic acid bacteria active during the fermentation of wheat silage. Europ. J. App!. Microbiol. Biotechnol. 13, 248-250 (1981) Rogosa, M.: Characters used in the classification of the lactobacilli. Int. J. System Bact. 20, 519-534 (1970) Rogosa, M., Sharpe, M. E.: An approach to the classification of the lactobacilli. J. App!. Bact. 22, 329-340 (1959) Schleifer, K. H.: Recent changes in the taxonomy of lactic acid bacteria. FEMS Microbiol. Rev. 46, 201-203 (1987) Schleifer, K. H., Kandler, 0.: Die Aminosauresequenz des Mureins von Streptococcus thermophilus und Streptococcus faecalis. Arch. Microbio!. 57, 335-364 (1967) Schleifer, K. H., Kandler, 0.: Peptidoglycan types of bacterial cell walls and their taxonomic implications. Bact. Rev. 36, 407-477 (1972) Seale, D. R.: Bacterial inoculants as silage additives. J. App!. Bact. Symp. Supp!. 1986, 9S-26S (1986) Sharpe, M. E.: Taxonomy of the lactobacilli. Dairy Sci. Abstr. 24, 109-118 (1962) Sharpe, M. E.: The Genus Lactobacillus. In: The Procaryotes. A Handbook on Habitats, Isolation, and Identificatin of Bacteria. (M. P. Starr, H. Stolp, H. G. Triiper, A. Balows, and H. G. Schlegel, eds.), Vo!' II, pp. 1653-1679. Berlin-HeidelbergNew York, Springer-Verlag 1981 Stetter, K. 0., Kandler, 0.: Untersuchung zur Entstehung von DL-Milchsaure bei Lactobacillen und Charakterisierung einer Milchsaureeracemase bei einigen Arten der Untergattung Streptobacterium. Arch. Mikrobio!. 94,221-247 (1973) Suhaimi, M., Bruyneel, B., Verstraete, W.: Ensilage of ammoniatreated straw in combination with whey by means of alkalineadapted lactic acid bacteria. J. App!. Bact. 63, 125-132 (1987a) Suhaimi, M., Bruyneel, B., Verstraete, W.: Enrichment of an alkaline-adapted association of streptococci and lactobacilli. J. App!. Microbio!. 63,117-123 (1987b) Teuber, M., Geis, A.: The Family Streptococcaceae (Nonmedical Aspects). In: The Procaryotes. A Handbook on Habitats, Isolation, and Identification of Bacteria (M. P. Starr, H. Stolp, H. G. Triiper, A. Balows and H. G. Schlegel, eds.), Vo!' II, pp. 1614-1630. Berlin-Heidelberg-New York, Springer-Verlag 1981 Wieringa, G. W.: Some factors influencing silage fermentation. Neth. J. Agric. Sci. 7, 237-241 (1960)
Professor Dr.]. Winter, Institut fiir Mikrobiologie, Universitat Regensburg, Universitatsstr. 31, D-8400 Regensburg
Lactobacillus graminis sp. nov., a New Species of Facultatively Heterofermentative Lactobacilli Surviving at Low pH in Grass Silage R. BECK!, N. WEISS 2, and]. WINTER3 1
2
3
Bayerische Landesanstalt rur Bodenkultur und Pflanzenbau, D-8000 Munchen 19, Federal Republic of Germany Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, D-3300 Braunschweig, Federal Republic of Germany Institut rur Mikrobiologie, Universitiit Regensburg, D-8400 Regensburg, Federal Republic of Germany Received February 10, 1988
Summary A group of 11 facultatively heterofermentative rod-shaped bacteria were isolated at a late fermentation stage of pre-wilted grass. These bacteria were found to differ from currently described lactobacilli in the spectrum of fermented sugars, in particular in their ability to ferment xylose. The isolates were similar to Lactobacillus curvatus and Lactobacillus sake with respect to D( -)- and L( +)- lactate formation, Rr values of the D- and L-LDH, G+C content of the DNA and the L-Lys-D-Asp murein type. Teichoic acid was lacking. DNA-DNA homology among the isolates was high, while low homology values were obtained with the type strains of L. curvatus and L. sake, indicating only a distant relationship. It is proposed that the isolates represent a new species named Lactobacillus graminis sp. nov.
Key words: Lactobacillus graminis sp. nov. - Lactobacilli - Taxonomy - DNA- DNA-homology - Grass silage - Population
Introduction The microbial ecology of fermenting vegetable material, including silages, and the use of bacterial inoculants as silage additives (starter cultures) has been reviewed recently (Seale, 1986; Daeschel et ai. 1987). The development of the flora of lactic acid bacteria during ensilage of different crops was found to be influenced mainly by the dry matter content of the crops and by the time of fermentation (Wieringa, 1960). The succession of single species has been studied during ensilage of wheat (Moon et aI., 1981) and, more recently, of ammonia-treated straw (Suhaimi et aI., 1987a). Straw fermentation required the enrichment of an alkaline-adapted association of streptococci and lactobacilli (Suhaimi et aI., 1987b). In earlier studies on the taxonomy of facultatively heterofermentative lactobacilli (as defined by Schleifer, 1987) from silage, few differences were noted between the isolates (Abo-Elnaga and Kandler, 1965a, b; Beck, 1965; Beck et aI., 1987a, b; Bucher, 1970). In this contribution we report the results of our studies on the physiology of the ensilage process of grass and the succession of lactobacilli during this process. A large number of lactobacilli
was isolated. One group of these from a late phase of the grass fermentation differed from currently described species and thus was studied in detail. These isolates are considered to belong to a new species of lactobacilli, for which the name Lactobacillus graminis sp. nov. is proposed. Material and Methods Organisms. The type strains of Lactobacillus curvatus (DSM 20019) and Lactobacillus sake (DSM 20017) were obtained from the Deutsche Sammlung von Mikroorganismen, D-3300 Braunschweig-Stockheim. Medium and growth conditions. Isolates from grass silage were obtained by repeated plating of diluted samples of silage juice on MRS-agar (DeMan et ai., 1960). Type strains and all isolates were cultured in MRS-medium (DeMan et ai., 1960) at 28°C. Stock cultures were kept in conservation agar stabs (Wieringa, 1960) at 4°C. Determination of the spectrum of fermented substrates. The ability of the isolates to ferment different sugars was tested ac-
280
R. Beck, N. Weiss, and J. Winter
cording to Rogosa and Sharpe (1959), Sharpe (1962) and Rogosa (1970). Sugar solutions were filter-sterilized and added to the sugar-free basal medium to give a final concentration of 1 gil. Disc electrophoresis.The electrophoretic mobility of the lactate dehydrogenase (LDH) was determined in polyacrylamide gels according to Maurer (1968), as modified by Stetter and Kandler (1973). Rabbit L-lactate dehydrogenase isoI served as a reference. Analysis of fermentation products. The configuration and the amount of lactic acid was determined enzymatically according to Horost (1970), using a test kit with either L- or D-lactate dehydrogenase. Acetic acid was determined with acetate kinase according to Ho/z and Bergmeyer (1970). The amount of ethanol was quantified using ethanol dehydrogenase and acetaldehyde dehydrogenase as described by Beutler and Michal (1977). Cell wall analysis. Cell walls were prepared and the murein components determined according to Schleifer and Kandler (1967, 1972). Teichoic acids were extracted from cell walls using the hydrofluoric acid (HF) method described by Fiedler et al. (1981). Sugar alcohols were separated as the corresponding alditol-acetate-derivatives (Albersheimer et al., 1967) by gas-liquid chromatography. Determination of the G+C content of the DNA and DNADNA homology studies. DNA was extracted and purified according to a modified method of Marmur (1961). The melting point (T M) of the DNA was determined according to Marmur and Doty (1962) and the G+C mol% was calculated using the equation of DeLey (1970). DNA-DNA hybridization experiments and the calculation of the % homology values were carried out as described by DeLey et al. (1970), Gillis et al. (1970) and Huss et al. (1983).
Results and Discussion Succession of lactobacilli and acid formation during the ensilage process of pre-wilted grass
Pre-wilted meadow grass with a dry matter content of 47.6% was chopped and ensilaged at 25°e in several laboratory silos of 11 capacity for 90 days. Two silos each were opened for chemical and microbiological analyses at different intervals. Lactic acid production reached the maximum after 30 days, while the maximum acetic acid concentration was reached after only 1 day (Fig. 1). The majority of the grass protein was degraded within one week, as judged from'ammonia production. Protein degradation was slow, but almost linearly proceeding for three months, while the maximum amount of lactic acid was reached after 1 month (Fig. 1). The number of total viable colony forming units (cfu,) was highest after 7 days (108/g) and declined to 107/g after 30 days and less than 106/g after 90 days (Fig. 1). To determine the succession of different species or groups of lactic acid bacteria (viable cells) during the ensilage process, samples were serially diluted and streaked on MRS-agar plates. After incubation at 28°e, 10 colonies of each distinguishable colony type from plates containing approximately 100 colonies, were inoculated into liquid medium. A rough classification of the isolates was performed by testing for the following features: morphology, colour of colonies, gas formation (with Durham tubes), growth at 15 and 45 °e, formation of D( -)-, L( +)- or DILlactate, NH3 production from arginine and by the spectrum of fermented sugars. Furthermore, the presence/abs-
:x:
a.
5.5
0.1 ~
5.0
Q052
,.,..,
:::; ::; 4 5
0
a.a,20 ~:g 15 ..... u dCj
Qj~
10
-.J
5
Uu «Cj
o X
0 01 3 7
8
-14
30
65
Time (dJ
•
i
7 ~ 6 '5 :J 0.0-
U
5 E' 90 •
Fig. 1. Kinetics of acetate-, D/L-lactate and ammonia formation and total number of viable population during ensilage of prewilted grass.
ence of m-DAP in the cell walls was analysed. The succession of Streptococcaceae and lactobacilli, tentatively identified according to Teuber and Geis (1981) and Sharpe (1981) is shown in Fig. 2. Initially streptococci (= lactococci; Schleifer, 1987), Lactobacillus coprophilus, Pediococcus pentosaceus and Leuconostoc sp. were predominant. Only when these organisms had lost viability, were other strains detected which could not be classified as strains of a known species of homofermentative lactobacilli, mainly due to their pentose utilization pattern. Upon further characterization these strains turned out as members of a new species, named Lactobacillus gram in is spec. nov. L. graminis strains may have been present in earlier stages of fermentation, but not in sufficiently high numbers to be detected. L. graminis comprised approximately 10% of the viable population after 30 days but up to 40% after 90 days. Together with Leuconostoc sp., Lactobacillus plantarum and P. pentosaceus, it appeared to survive longer in the silage than other lactobacilli. Eleven strains of L. graminis were isolated from samples drawn at day 30 and day 90. Five of these strains were further characterized.
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Fig. 2. Population dynamics of lactic acid bacteria during ensilage of pre-wilted grass.
Lactobacillus graminis sp. nov.
Characteristics of Lactobacillus graminis sp. nov.
All isolates, identified as L. graminis formed smooth, non-pigmented round colonies of 1-2 mm in diameter. In liquid cultures the rod-shaped, slightly curved cells, 0.7-1 by 1.5-2 [.Lm in size, occured singly, in pairs or in chains up to 10 [.Lm (Fig. 3) A flocculant sediment was formed after 3 days of growth. Cells stained Gram-positive. A comparison of physiological features of Lactobacillus graminis isolates with those of other lactobacilli is shown in Table 1. The L-Lys-D-Asp murein type (4 isolates tested), the G+C content of the DNA (5 isolates tested, 41-43 mol% G+C) and the electrophoretic mobility of the lactate dehydrogenases of 5 isolates of L. graminis were identical. On the basis of these properties L. graminis isolates could not be differentiated from L. curvatus and L. sake. However, strains of L. graminis differed from L. curvatus and L. sake in several other characteristics, e. g. the ability to ferment maltose, ribose and xylose (Table 1). The inability to ferment xylose is considered to be an important constant feature of the facultatively heterofermentative lactobacilli (Schleifer, 1987). Fermentation balances
Glucose and xylose fermentation by two of the eleven isolates of L. graminis was analysed. One mol of glucose was almost stoichiometrically fermented to 2 mol D/Llactate. Only a small amount of acetate was formed (Table 2). One mol xylose was fermented to 1 mol D/L-Iactate and 1 mol acetate. No ethanol was produced. A similar fermentation balance was found for arabinose and ribose fermentation by L. plantarum (Table 2). In very young cultures (growth time 6 h) of both L. graminis strains tested, L-Iactate predominated over (only traces of) Dlactate, but an almost racemic mixture was found in stationary cultures.
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++1+++1 +++++++ ++1++++ +++++++ ++1++++ ++-0++++ 1+ 1+ + + 1 I 1 I 1 I I ++-0++++
281
282
R. Beck, N. Weiss, and J. Winter
Species
Substrate
L-Lactate
V
Table 2. Fermentation balance of glucose, xylose, ribose and arabinose by resting cells of L. graminis and L. plantarum
Acetate
a
b
a
b
a
b
31,1 11,1
0,89 0,36
3,3 29,6
0,095 0,95
0,4 0,15
0,4 18,4
0,065 0,98
0,71 0,37 0,33
0,2 28,8 21,3
0,007 0,72 0,665
L. graminis G30 (5)
Glucose Xylose
35 31
37,5 21,4
1.07 0,69
L. graminis G90 (1)
Glucose Xylose
6,5 18,7
10,1 12,8
1,55 0,68
L. plantarum
Glucose Arabinose Ribose
28,3 40,0 32,0
43,5 26,4 21,4
1,54 0,66 0,67
R30 (19)
D-Lactate
2.6 2,85 20,2 14,7 10,6
V = Fermented substrate in mmolJl a = produced acid in mmolll (total) b = produced acid in mmolJl per fermented mmolJl substrate
Genomic characteristics L. graminis strains
Gram-positive, non-motile, slightly curved rods with
The G+C content of the DNA of L. graminis strain G 90(1) was 42 mol%. The G+C content for 4 other L. graminis strains varied between 41-43 mol%. DNA-DNA hybridisation studies showed a high homology (90-100%) among the five strains of L. graminis (Table 3) tested, but low values between the L. graminis strains and the type strains of L. curvatus and L. sake (20% and 40%, respectively). These low hybridisation values indicate only a distant relationship between L. gram in is, L. curvatus and L. sake and, together with differences in the spectrum of fermented sugars, define the new species L. graminis. Description of Lactobacillus graminis sp. nov. Lactobacillus graminis sp. nov., gra'mi.nis. L. n. gramen, grass, graminis of grass.
r~)Unde? en?s, g~nerally 0.7:-1 by 1.5-2 f.lm, occuring
smgly, m paIrS or m short chams, slightly curved. Colonies smooth, round, non-pigmented. Flocculant sediment after three days of growth in MRS-broth. Facultatively heterofermentative. Produce exclusively DIL-Iactate from glucose and D/L-Iactate and acetate from xylose. Mic.roaerophilic. No growth at 45°C, growth at 15 0c. Opnmal temperature 30-35°C. Fermentation of carbohydrates and other characteristics are as given in Table 1. The peptidoglycan is of the L-Lys-D-Asp type. Cell wall teichoic acid not detectable. The G+ C content of the DNA is in the range 41-43 mol%, for strain G90(1) 42 mol% (TM ). Habitat: grass silage. Type strain: Lactobacillus graminis strain G90(1) DSM 20719. '
Table 3. DNA-DNA-hybridisation (% homology) of L. graminis strains and related lactobacilli
L. sake 27/85
L. graminis strains G30(13) G90(8)
L. sake
L. curvatus
20017
20019
G90(1)
78
41.5
27.3
40
39.4
32.8
30.6
28
36
37.2
48
35
G30(5)
G90(3)
L. sake
27/85
100
L. sake
20017
100
L. curvatus
20019
100
L. graminis
G90(1) G30(5) G90(8) ".. )lot -4etermined
100
100 100
94.5 100 100
94.8 96.4 91.5
97.2
Lactobacillus graminis sp. nov.
References Abo-Elnaga, ]. G., Kandler, 0.: Zur Taxonomie der Gattung Lactobacillus Beijerinck. I: Das Subgenus Streptobacterium Orla-Jensen. Zbl. Bakt., II Abt. 119, 1-36 (1965a) Abo-Elnaga, ]. G., Kandler, 0.: Zur Taxonomie der Gattung Lactobacillus Beijerinck. II: Das Subgenus Betabacterium Orla-Jensen. Zbl. Bakt., II Abt. 119, 177-129 (1965b) Albersheim, P., Nevois, D.]., Englis, P. D., Karr, A.: A method for the analysis of sugars in plant cell wall polysaccharides by gas-liquid chromatography. Carbohydr. Res. 5, 340-345 (1967) Beck, Th.: Untersuchungen iiber die Okologie und Physiologie der Giirfuttermikroflora. Landw. Forschung 19, 3-11 (1965) Beck, R., Grop, F., Beck, Th.: Untersuchungen zur Kenntnis der Gatfuttermikroflora, I. Mitteilung: Die Zusammensetzung der Milchsaurebakterienpopulation bei der Vergarung von Deutschem Weidelgras und Rotklee. Z. Das wirtschaftseigene Futter 33, 13-33 (1987a) Beck, Th., Grop, F., Beck, R.: Untersuchungen zur Kenntnis der Garfuttermikroflora, II. Mitteilung: Milchsaurebakterien von Silagen unterschiedlicher Qualitat aus Praxissilagen. Z. Das wirtschaftseigene Futter 33, 34-43 (1987b) Beutler, H. 0., Michal, G.: Neue Methode zur enzymatischen Bestimmung von Athanol in Lebensmitteln. Fresenius J. Ana!. Chern. 284,113-117 (1977) Bucher, E.: Beitriige zur Mikrobiologie der Silagegarung und der Garfutterstabilitat. Dissertation, Universitat Miinchen 1970 Daeschel, M. A., Anderson, R. E., Fleming, H. P.: Microbial ecology of fermenting plant materia!' FEMS Microbio!. Rev. 46, 357-367 (1987) DeLey, ].: Reexamination oJ the association between melting point, buoyant density, and chemical base composition of deoxyribonucleic acid. J. Bact. 101, 738-754 (1970) DeMan, ]. C., Rogosa, M., Sharpe, M. E.: A medium for the cultivation of lactobacilli. J. App!. Bact. 23, 130-135 (1960) Fiedler, F., Schaffler, M. ]., Stackebrandt, E.: Biochemical and nucleic acid hybridisation studies on Brevibacterium linens and related strains. Arch. Microbiol. 129, 85-91 (1981) Gillis, M., De Ley, ]., DeCleene, M.: The determination of molecular weight of bacterial genome DNA from renaturation rates. Europ. J. Biochem. 12, 143-153 (1970) Holz, G., Bergmeyer, H. U.: Acetatbestimmung. In: Methoden der enzymatischen Analyse (H. U. Bermeyer, ed.). Weinheim, Verlag Chemie 1970 Horost, H. ].: L-Lactatbestimmung. In: Methoden der enzymatischen Analyse (H. U. Bergmeyer, ed.), pp.266-270. Weinheim, Verlag Chemie 1970 Huss, V. A . R., Fest, R., Schleifer, K. H.: Studies on the spectrophotometric determination of DNA hybridization from renaturation rates. System. Appl. Microbiol. 4, 184-192 (1983) Kandler, 0., Weiss, N.: Genus Lactobacillus. In: Bergey's Manual of Systematic Bacteriology, Vol. II (P. H. A. Sneath and]. G.
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Holt, eds.), p. 1208. Baltimore, The Williams and Wilkens Co. 1986 Marmur, ].: A procedure for the isolation of DNA from microorganisms. J. Molec. BioI. 3, 208-218 (1961) Marmur, ]., Doty, P.: Determination of the base composition of deoxyribonucleic acid from its thermal denaturation temperature. J. Molec. Bio!. 5, 109-118 (1962) Maurer, H. R.: Disk-Elektrophorese. Berlin, Walter de Gruyter Verlag 1968 Moon, N. ]., Moon, L. c., Ely, L. 0., Parker, ]. A.: Lactic acid bacteria active during the fermentation of wheat silage. Europ. J. App!. Microbiol. Biotechnol. 13, 248-250 (1981) Rogosa, M.: Characters used in the classification of the lactobacilli. Int. J. System Bact. 20, 519-534 (1970) Rogosa, M., Sharpe, M. E.: An approach to the classification of the lactobacilli. J. App!. Bact. 22, 329-340 (1959) Schleifer, K. H.: Recent changes in the taxonomy of lactic acid bacteria. FEMS Microbiol. Rev. 46, 201-203 (1987) Schleifer, K. H., Kandler, 0.: Die Aminosauresequenz des Mureins von Streptococcus thermophilus und Streptococcus faecalis. Arch. Microbio!. 57, 335-364 (1967) Schleifer, K. H., Kandler, 0.: Peptidoglycan types of bacterial cell walls and their taxonomic implications. Bact. Rev. 36, 407-477 (1972) Seale, D. R.: Bacterial inoculants as silage additives. J. App!. Bact. Symp. Supp!. 1986, 9S-26S (1986) Sharpe, M. E.: Taxonomy of the lactobacilli. Dairy Sci. Abstr. 24, 109-118 (1962) Sharpe, M. E.: The Genus Lactobacillus. In: The Procaryotes. A Handbook on Habitats, Isolation, and Identificatin of Bacteria. (M. P. Starr, H. Stolp, H. G. Triiper, A. Balows, and H. G. Schlegel, eds.), Vo!' II, pp. 1653-1679. Berlin-HeidelbergNew York, Springer-Verlag 1981 Stetter, K. 0., Kandler, 0.: Untersuchung zur Entstehung von DL-Milchsaure bei Lactobacillen und Charakterisierung einer Milchsaureeracemase bei einigen Arten der Untergattung Streptobacterium. Arch. Mikrobio!. 94,221-247 (1973) Suhaimi, M., Bruyneel, B., Verstraete, W.: Ensilage of ammoniatreated straw in combination with whey by means of alkalineadapted lactic acid bacteria. J. App!. Bact. 63, 125-132 (1987a) Suhaimi, M., Bruyneel, B., Verstraete, W.: Enrichment of an alkaline-adapted association of streptococci and lactobacilli. J. App!. Microbio!. 63,117-123 (1987b) Teuber, M., Geis, A.: The Family Streptococcaceae (Nonmedical Aspects). In: The Procaryotes. A Handbook on Habitats, Isolation, and Identification of Bacteria (M. P. Starr, H. Stolp, H. G. Triiper, A. Balows and H. G. Schlegel, eds.), Vo!' II, pp. 1614-1630. Berlin-Heidelberg-New York, Springer-Verlag 1981 Wieringa, G. W.: Some factors influencing silage fermentation. Neth. J. Agric. Sci. 7, 237-241 (1960)
Professor Dr.]. Winter, Institut fiir Mikrobiologie, Universitat Regensburg, Universitatsstr. 31, D-8400 Regensburg