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Lactobacillus bifermentans sp. nov., nom. rev., an Organism Forming CO2 and H2 from Lactic Acid
System. Appl. Microbiol. 4, 408-412 (1983) BotanischesInstitut der Universitat Miinchen, 8000Miinchen 19 2Deutsche Sammlungvon Mikroorganismen, Gesellschaft fur Biotechnologische Forschung mbH, 3300 Braunschwcig-Stockheirn, Federal Republic of Germany
1
Lactobacillus bifermentans sp. nov., nom. rev., an Organism Forming CO 2 and H 2 from Lactic Acid OTTO KANDLER\ ULRICH SCHILLINGER\ and NORBERT WEISS 2 Received May 15, 1983
Summary Lactobacillus "bifermentans", an organism causing small cracks by gas formation in Edam and Gouda cheeses, has beenisolated and describedby Pette and Van Beynum (1943). In this paper the ability of Lactobacillus "bifermentans" to carry out homolactic fermentation at high glucose concentrations, as well as its ability to ferment lactate at pH > 4.0 to acetic acid, ethanol, traces of propionic acid, CO 2 and H 2 is confirmed. In addition, several biochemical characteristics are determined. In spite of the fact that the organism is able to form H 2 , which is untypical of lactobacilli, it is consideredto belong to the genus Lactobacillus. Hence, the original name Lactobacillus "bifermentans" is revived.
Key words: Lactobacillus biiermentans - Lactate Degradation - H 2 Formation Cheese blowing Boekhout and Ott de Vries (1918) isolated bacteria from Edam and Gouda cheeses which showed the defect called "Boekelscheuren": small cracks formed in addition to the desired round holes, thus devaluing the cheeses markedly (Boekhout and Ott de Vries, 1910). These bacteria fermented not only sugars to lactic acid, but also lactic acid to acetic acid, ethanol, CO 2 and Hz. The formation of gas was considered to be the cause of crack formation (Boekelscheuren). In more detailed studies, Pette and van Beynum (1943) confirmed the unusual fermentation pattern of bacteria, again isolated from Dutch cheeses exhibiting the defect "Boekelscheuren". They found homolactic fermentation when glucose was given in excess. Lactic acid was, however, fermented according to equation (I), when the medium, acidified by lactic acid fermentation, was neutralized, or, when the organism was grown on limiting amounts of glucose, or with lactate as the only fermentable substrate. (I) 2 lactic acid -+ acetic acid + ethanol + 2 CO 2 + 2 Hz In most fermentations, the amount of Hz was lower than that expected according to equation (I) and small amounts of propionic acid were formed in addition to acetic acid and ethanol.
Lactobacillus bifermentans sp. nov., nom. rev.
409
The organism was named Lactobacillus "biierrneruans't! (Pette and van Beynum, 1943) although the formation of free H 2 speaks against its inclusion in the genus Lactobacillus. Thus L. "biiermentans" is listed among the "species incertae sedis" in the 8th edition of Bergey's Manual and its taxonomic position within the genus Lactobacillus has been questioned. It is not contained in the "Approved Lists of Bacterial Names" (Skerman et al., 1980). However, Stackebrandt et al., (1983) studying the genealogical relationships between lactic acid bacteria by comparing the RNase T1 - resistant oligo-nucleotides of the 16S ribosomal RNA (SAB values), found L. "bifermentans" to be a member of the Lactobacillus cluster and specifically related to Lactobacillus casei. The latter is also known to ferment glucose to acetic acid and ethanol under glucose limitation. The C-1 compound released by L.casei is, however, formic acid instead of CO 2 and no H 2 is formed (De Vries et al., 1970). We have reinvestigated the characteristics of L. "bifermentans" DSM 20003 derived from strain N 2 isolated by Pette and Beynum (1943). Morphology (Fig. 1), the range of fermented saccharides (Table 1) and the fermentation products were the same as those described by Pette and Beynum (1943). In addition, D- and L-lactic acid dehydrogenases could be found upon polyacrylamide gel electrophoresis, using D- or L-lactate as substrates (according to the methods of Stetter and Kandler, 1973). The migration distance of the D- and L-enzyme relative to that of isoenzyme I of the rabbit L-lactic acid dehydrogenase was 1.1. and 1.2., respectively. Neither of the two enzymes was activated by FDP and Mn 2+ as is the case with the allosteric enzyme of L. casei (Hensel et al., 1977) and related species. In MRS medium (De Man et al., 1960) with 1% glucose L. "bifermentans" forms almost exclusively DL-lactic acid (Table 2). Mainly Acetate, ethanol, CO 2 , H 2 and small amounts of propionic acid were, however, formed at glucose concentrations of < 1.0% and neutralization with phosphatebuffer (Pette and Beynum, 1943), or when the organism was grown in MRS medium or pepton-yeast extract broth with DL-lactate as the only or main fermentable substrate (Table 2). After several days of adaptation, D-ribose was fermented to DL-lactic acid, acetic acid, ethanol, CO 2 and H 2 (Table 2). Further studies on pentose fermentation are necessary to elucidate its mechanism. Cell wall analyses (d. Schleifer and Kandler 1967, 1972) revealed the presence of murein of the Lys-Asp type, the most common type in lactobacilli. Glucose and galactose were found in cell wall hydrolysates (2N HCl, 2 h, 100°C) by gas chromatography. Neither glycerol nor ribitol could be detected by gas chromatography, and the phosphate content of the isolated cell walls was below 1%. Thus teichoic acid is not present. The GC content of DNA calculated from the melting point (De Ley, 1971) is 45 mol%. DNA of E. coli B was used as reference. DNA-DNA hybridization of L. "biiermentans" with all known species of heterofermentative lactic acid bacteria (Leuconostoc and beta bacteria) using filterbound and 3H-thymidine labeled DNA (Vescovo et al., 1979; Schillinger et al., 1983) showed no significant homology with any of these species. Thus it seems justified to consider the organism as a well defined species and to revive the original name. Description of Lactobacillus bilermentans sp. nov., nom. rev. (L. bi.ler.men'tans. L. pref. bis twice; L. part. adj. fermentans leavening; M.L. adj. biiermentans doubly fermenting). 1 Names between quotation marks are not containedin the "Approved Lists of Bacterial Names".
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= fermented (+) = delayed fermentation, dye reduction = not determined * = according to Pette and Van Beynum (1943)
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Lactobacillus bif ermentans sp. nov., nom. rev.
411
Fig. 1. Phase cont rast micrograph of 1. biierm entans grow n in M RS medium. T able 2. Ferment ation produ cts of 1. " bijermentans" Substrates a (flmol/ml) D-glucose 50 D-ribo se 67d Dt.-Iacrare 136
Dl. -Lactare" 2d 6d 69 32 69
74 13.5 61
Products in the fermented medium (flmol/ml) H2c C0 2 c Acetate" Ethanol" 6d 2d 2d 6d 2d 2d 6d 6d 6 38 30
6 55 36
9 25.0 32
9 58 43
0.9 19 31
1.0 18 43
7.0 23 40
8.0 62 48
Traces of prop ionate were present in all samples. a Th e substrates were added to 20 ml MRS medium withou t glucose and acetate in 120 ml stoppered serum bottl es ; N 2 in the gas phase ; incub ati on without shaking at 30 DC for 2 or 6 days. b Analysis was performed enzymatically according to Bergm eyer (1 970). c Analysis was perfor med by gaschrornarography. d Ribose adapted cells were used for inoculation.
Gram -positive, non-sporeforming short irregular rods with rounded, often tapered end s, generally 0.5-1.0 by 1.5-2.0 [1m , occurr ing singly, in pairs or short irregular chains often forming lumps (Fig.1). Non-mot ile. Colonies smooth and convex, greyish white, glistening. Surface growth is enhanced by anerobiosis. Hornoferment ative with production of DL-lactic acid in media containing > 1.0% ferment able hexoses. Lactic acid is fermented to acetic acid, ethanol, traces of propionic acid, CO 2 and H 2 at pH > 4.0. Growth at 15°C but not at 42 °C. Optimum growth temperature 30 °C. Th e fermented saccharides are listed in Table 1. Arginine is not hydrol yzed. Nitrate is not reduced. Catalase and benzidine tests negat ive. Indol, H 2S, lipase and urease not prod uced. Murein is of the Lys-Asp type. Cell wall cont ains no teicho ic acid.
412
a. Kandler,
U. Schillinger, and N. Weiss
The G + C content of the DNA is 45%. (Tm) Causes blowing (small cracks: boekelscheuren) in Edam and Gouda cheeses. Type strain: DSM 20003 (subculture of strain N 2 isolated by Pette and Van Beynum, 1943). Addentum at proof reading After the manuscript had been finished Dr. R. M. Kroppenstedt kindly supplied us with the following additional information: L. bifermentans does not contain menaquinones or ubiquinones. Cellular fatty acids are of the straight-chain saturated, mono-unsaturated and cyclopropane type. No branched is or anteiso fatty acids were detected. These results confirme the classification of L. bifermentans within the genus Lactobacillus.
References Bergmeyer, H. V.: Methoden der enzymatischen Analyse. 2. Auf!. Weinheim, Verlag Chernie, 1970 Boekhout, F. W. J., Ott de Vries, J.J.: Uber zwei Kasefehler in Edamer Kase, Zbl. Bakt., II. Abt. 28, 98-111 (1910) Boekhout, F. W.I., Ott de Vries, I.J.: Die normale Gasbildung im Edamer und Gouda Kase, Zbl. Bakt., ILAbt. 48, 130-139 (1918) Hensel, R., Mayr, V., Stetter, K. O.; Kandler, a.: Comparative studies of lactic acid dehydrogenases in lactic acid bacteria. L Purification and kinetics of the allosteric L-Iactic acid dehydrogenase from Lactobacillus casei ssp. casei and Lactobacillus curvatus. Arch. Microbiol. 112, 81-93 (1977) De Ley, I.: Hybridisation of DNA. Meth. Microbiol. SA, 311-314 (1971) De Man, J.R., Rogosa, M., Sharpe, M.E.: A medium for the cultivation of lactobacilli. J. appl. Bact. 23, 130-135 (1960) Pette, J. W., Van Beynum, I.: Boekelscheurbacterien Rijkslandbauwproefstation te hoorn. Versl. Landbouwk Onderz, 49 C, 315-346 (1943) Schillinger, V., Holzapfel, W., Weiss, N., Kandler, 0.: DNA/DNA homology between the type strains of the genus Leuconostoc and heterofermentative lactobacilli. System. Appl. Microbiol. 4 (in press, 1983) Schleifer, K. H., Kandler, 0.: Zur chemischen Zusammensetzung der Zellwand der Streptococcen. L Die Arninosauresequenz des Mureins von Streptococcus thermophilus und Streptococcus faecalis. Arch. Microbiol. 57, 335-364 (1967) Schleifer, K. H., Kandler, a.:Peptidoglycan types of bacterial cell walls and their taxonomic implications. Bact. Rev. 36, 407-477 (1972) Sherman, V.B.D., McGowan, V., Sneath, P.H.A.: Approved Lists of Bacterial Names. Int. J. system. Bact. 30, 224-420 (1980) Stackebrandt, E., Fowler, V.j., Woese, C. R.: A Phylogenetic Analysis of lactobacilli, Pediococcus pentosaceus and Leuconostoc mesenteroides. System. Appl. Microbiol. 4, (in press, 1983) Stetter, K.O., Kandler, 0.: Untersuchung zur Entstehung von Dl.-Milchsaure bei Lactobacillen und Charakterisierung einer Milchsaureracernase bei einigen Arten der Untergattung Streptobacterium. Arch. Microbiol. 94, 221-247 (1973) Vescovo, M., Dellaglio, F., Bottazzi, V., Sarra, P. G.: Deoxyribonucleic acid homology among Lactobacillus species of the subgenus Betabacterium aria-Jensen. Microbiologica 2, 317-330 (1979) De Vries, W., Kapteijn, W.M. C,; Van der Beek, E. G.: Molar growth yields and fermentation balances of Lactobacillus casei L 3 in batch cultures and in continous cultures. J. gen. Microbial. 63, 333-345 (1970) Professor Dr. Otto Kandler, Botanisches Institut der Univ., Menzinger Str. 67, D-8000 Miinchen 19
1
Lactobacillus bifermentans sp. nov., nom. rev., an Organism Forming CO 2 and H 2 from Lactic Acid OTTO KANDLER\ ULRICH SCHILLINGER\ and NORBERT WEISS 2 Received May 15, 1983
Summary Lactobacillus "bifermentans", an organism causing small cracks by gas formation in Edam and Gouda cheeses, has beenisolated and describedby Pette and Van Beynum (1943). In this paper the ability of Lactobacillus "bifermentans" to carry out homolactic fermentation at high glucose concentrations, as well as its ability to ferment lactate at pH > 4.0 to acetic acid, ethanol, traces of propionic acid, CO 2 and H 2 is confirmed. In addition, several biochemical characteristics are determined. In spite of the fact that the organism is able to form H 2 , which is untypical of lactobacilli, it is consideredto belong to the genus Lactobacillus. Hence, the original name Lactobacillus "bifermentans" is revived.
Key words: Lactobacillus biiermentans - Lactate Degradation - H 2 Formation Cheese blowing Boekhout and Ott de Vries (1918) isolated bacteria from Edam and Gouda cheeses which showed the defect called "Boekelscheuren": small cracks formed in addition to the desired round holes, thus devaluing the cheeses markedly (Boekhout and Ott de Vries, 1910). These bacteria fermented not only sugars to lactic acid, but also lactic acid to acetic acid, ethanol, CO 2 and Hz. The formation of gas was considered to be the cause of crack formation (Boekelscheuren). In more detailed studies, Pette and van Beynum (1943) confirmed the unusual fermentation pattern of bacteria, again isolated from Dutch cheeses exhibiting the defect "Boekelscheuren". They found homolactic fermentation when glucose was given in excess. Lactic acid was, however, fermented according to equation (I), when the medium, acidified by lactic acid fermentation, was neutralized, or, when the organism was grown on limiting amounts of glucose, or with lactate as the only fermentable substrate. (I) 2 lactic acid -+ acetic acid + ethanol + 2 CO 2 + 2 Hz In most fermentations, the amount of Hz was lower than that expected according to equation (I) and small amounts of propionic acid were formed in addition to acetic acid and ethanol.
Lactobacillus bifermentans sp. nov., nom. rev.
409
The organism was named Lactobacillus "biierrneruans't! (Pette and van Beynum, 1943) although the formation of free H 2 speaks against its inclusion in the genus Lactobacillus. Thus L. "biiermentans" is listed among the "species incertae sedis" in the 8th edition of Bergey's Manual and its taxonomic position within the genus Lactobacillus has been questioned. It is not contained in the "Approved Lists of Bacterial Names" (Skerman et al., 1980). However, Stackebrandt et al., (1983) studying the genealogical relationships between lactic acid bacteria by comparing the RNase T1 - resistant oligo-nucleotides of the 16S ribosomal RNA (SAB values), found L. "bifermentans" to be a member of the Lactobacillus cluster and specifically related to Lactobacillus casei. The latter is also known to ferment glucose to acetic acid and ethanol under glucose limitation. The C-1 compound released by L.casei is, however, formic acid instead of CO 2 and no H 2 is formed (De Vries et al., 1970). We have reinvestigated the characteristics of L. "bifermentans" DSM 20003 derived from strain N 2 isolated by Pette and Beynum (1943). Morphology (Fig. 1), the range of fermented saccharides (Table 1) and the fermentation products were the same as those described by Pette and Beynum (1943). In addition, D- and L-lactic acid dehydrogenases could be found upon polyacrylamide gel electrophoresis, using D- or L-lactate as substrates (according to the methods of Stetter and Kandler, 1973). The migration distance of the D- and L-enzyme relative to that of isoenzyme I of the rabbit L-lactic acid dehydrogenase was 1.1. and 1.2., respectively. Neither of the two enzymes was activated by FDP and Mn 2+ as is the case with the allosteric enzyme of L. casei (Hensel et al., 1977) and related species. In MRS medium (De Man et al., 1960) with 1% glucose L. "bifermentans" forms almost exclusively DL-lactic acid (Table 2). Mainly Acetate, ethanol, CO 2 , H 2 and small amounts of propionic acid were, however, formed at glucose concentrations of < 1.0% and neutralization with phosphatebuffer (Pette and Beynum, 1943), or when the organism was grown in MRS medium or pepton-yeast extract broth with DL-lactate as the only or main fermentable substrate (Table 2). After several days of adaptation, D-ribose was fermented to DL-lactic acid, acetic acid, ethanol, CO 2 and H 2 (Table 2). Further studies on pentose fermentation are necessary to elucidate its mechanism. Cell wall analyses (d. Schleifer and Kandler 1967, 1972) revealed the presence of murein of the Lys-Asp type, the most common type in lactobacilli. Glucose and galactose were found in cell wall hydrolysates (2N HCl, 2 h, 100°C) by gas chromatography. Neither glycerol nor ribitol could be detected by gas chromatography, and the phosphate content of the isolated cell walls was below 1%. Thus teichoic acid is not present. The GC content of DNA calculated from the melting point (De Ley, 1971) is 45 mol%. DNA of E. coli B was used as reference. DNA-DNA hybridization of L. "biiermentans" with all known species of heterofermentative lactic acid bacteria (Leuconostoc and beta bacteria) using filterbound and 3H-thymidine labeled DNA (Vescovo et al., 1979; Schillinger et al., 1983) showed no significant homology with any of these species. Thus it seems justified to consider the organism as a well defined species and to revive the original name. Description of Lactobacillus bilermentans sp. nov., nom. rev. (L. bi.ler.men'tans. L. pref. bis twice; L. part. adj. fermentans leavening; M.L. adj. biiermentans doubly fermenting). 1 Names between quotation marks are not containedin the "Approved Lists of Bacterial Names".
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+
- = not fermented
DSM 20003 N*2; E'\; C,\ 12* A"s
Strains
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Table 1. Range of sugars fermented by strains of L. "bifermentans"
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Lactobacillus bif ermentans sp. nov., nom. rev.
411
Fig. 1. Phase cont rast micrograph of 1. biierm entans grow n in M RS medium. T able 2. Ferment ation produ cts of 1. " bijermentans" Substrates a (flmol/ml) D-glucose 50 D-ribo se 67d Dt.-Iacrare 136
Dl. -Lactare" 2d 6d 69 32 69
74 13.5 61
Products in the fermented medium (flmol/ml) H2c C0 2 c Acetate" Ethanol" 6d 2d 2d 6d 2d 2d 6d 6d 6 38 30
6 55 36
9 25.0 32
9 58 43
0.9 19 31
1.0 18 43
7.0 23 40
8.0 62 48
Traces of prop ionate were present in all samples. a Th e substrates were added to 20 ml MRS medium withou t glucose and acetate in 120 ml stoppered serum bottl es ; N 2 in the gas phase ; incub ati on without shaking at 30 DC for 2 or 6 days. b Analysis was performed enzymatically according to Bergm eyer (1 970). c Analysis was perfor med by gaschrornarography. d Ribose adapted cells were used for inoculation.
Gram -positive, non-sporeforming short irregular rods with rounded, often tapered end s, generally 0.5-1.0 by 1.5-2.0 [1m , occurr ing singly, in pairs or short irregular chains often forming lumps (Fig.1). Non-mot ile. Colonies smooth and convex, greyish white, glistening. Surface growth is enhanced by anerobiosis. Hornoferment ative with production of DL-lactic acid in media containing > 1.0% ferment able hexoses. Lactic acid is fermented to acetic acid, ethanol, traces of propionic acid, CO 2 and H 2 at pH > 4.0. Growth at 15°C but not at 42 °C. Optimum growth temperature 30 °C. Th e fermented saccharides are listed in Table 1. Arginine is not hydrol yzed. Nitrate is not reduced. Catalase and benzidine tests negat ive. Indol, H 2S, lipase and urease not prod uced. Murein is of the Lys-Asp type. Cell wall cont ains no teicho ic acid.
412
a. Kandler,
U. Schillinger, and N. Weiss
The G + C content of the DNA is 45%. (Tm) Causes blowing (small cracks: boekelscheuren) in Edam and Gouda cheeses. Type strain: DSM 20003 (subculture of strain N 2 isolated by Pette and Van Beynum, 1943). Addentum at proof reading After the manuscript had been finished Dr. R. M. Kroppenstedt kindly supplied us with the following additional information: L. bifermentans does not contain menaquinones or ubiquinones. Cellular fatty acids are of the straight-chain saturated, mono-unsaturated and cyclopropane type. No branched is or anteiso fatty acids were detected. These results confirme the classification of L. bifermentans within the genus Lactobacillus.
References Bergmeyer, H. V.: Methoden der enzymatischen Analyse. 2. Auf!. Weinheim, Verlag Chernie, 1970 Boekhout, F. W. J., Ott de Vries, J.J.: Uber zwei Kasefehler in Edamer Kase, Zbl. Bakt., II. Abt. 28, 98-111 (1910) Boekhout, F. W.I., Ott de Vries, I.J.: Die normale Gasbildung im Edamer und Gouda Kase, Zbl. Bakt., ILAbt. 48, 130-139 (1918) Hensel, R., Mayr, V., Stetter, K. O.; Kandler, a.: Comparative studies of lactic acid dehydrogenases in lactic acid bacteria. L Purification and kinetics of the allosteric L-Iactic acid dehydrogenase from Lactobacillus casei ssp. casei and Lactobacillus curvatus. Arch. Microbiol. 112, 81-93 (1977) De Ley, I.: Hybridisation of DNA. Meth. Microbiol. SA, 311-314 (1971) De Man, J.R., Rogosa, M., Sharpe, M.E.: A medium for the cultivation of lactobacilli. J. appl. Bact. 23, 130-135 (1960) Pette, J. W., Van Beynum, I.: Boekelscheurbacterien Rijkslandbauwproefstation te hoorn. Versl. Landbouwk Onderz, 49 C, 315-346 (1943) Schillinger, V., Holzapfel, W., Weiss, N., Kandler, 0.: DNA/DNA homology between the type strains of the genus Leuconostoc and heterofermentative lactobacilli. System. Appl. Microbiol. 4 (in press, 1983) Schleifer, K. H., Kandler, 0.: Zur chemischen Zusammensetzung der Zellwand der Streptococcen. L Die Arninosauresequenz des Mureins von Streptococcus thermophilus und Streptococcus faecalis. Arch. Microbiol. 57, 335-364 (1967) Schleifer, K. H., Kandler, a.:Peptidoglycan types of bacterial cell walls and their taxonomic implications. Bact. Rev. 36, 407-477 (1972) Sherman, V.B.D., McGowan, V., Sneath, P.H.A.: Approved Lists of Bacterial Names. Int. J. system. Bact. 30, 224-420 (1980) Stackebrandt, E., Fowler, V.j., Woese, C. R.: A Phylogenetic Analysis of lactobacilli, Pediococcus pentosaceus and Leuconostoc mesenteroides. System. Appl. Microbiol. 4, (in press, 1983) Stetter, K.O., Kandler, 0.: Untersuchung zur Entstehung von Dl.-Milchsaure bei Lactobacillen und Charakterisierung einer Milchsaureracernase bei einigen Arten der Untergattung Streptobacterium. Arch. Microbiol. 94, 221-247 (1973) Vescovo, M., Dellaglio, F., Bottazzi, V., Sarra, P. G.: Deoxyribonucleic acid homology among Lactobacillus species of the subgenus Betabacterium aria-Jensen. Microbiologica 2, 317-330 (1979) De Vries, W., Kapteijn, W.M. C,; Van der Beek, E. G.: Molar growth yields and fermentation balances of Lactobacillus casei L 3 in batch cultures and in continous cultures. J. gen. Microbial. 63, 333-345 (1970) Professor Dr. Otto Kandler, Botanisches Institut der Univ., Menzinger Str. 67, D-8000 Miinchen 19