Taxonomy of Acetic Acid Bacteria

Utilized for Vinegar Fermentation

Yuzo YAMADA

BIOTEC Culture Collection, National Center for Genetic Engineeringand Biotechnology, National Science and Technology DevelopmentAgency, Pathumthani 12120, THAILAND

The acetic acid bacteria that are nowadays accommodated in the familyAcetobacteraceae Gillis & De Ley 1980, the α-ProteobacteriaStackebrandt et al. 1988 are classified in the following six genera:Acetobacter Beijerinck 1898, Gluconobacter Asai 1935, AcidomonasUrakami et al. 1989, Gluconacetobacter Yamada et al. 1997, AsaiaYamada et al. 2000 and Kozakia Lisdiyanti et al. 2002.

Of the six genera, the acetic acid bacteria that are utilized for vinegarfermentation are distributed mainly in the genera Acetobacter andGluconacetobacter. In the latter genus, the vinegar-producing organismsare restricted phylogenetically to the sublineage B, which includesGluconacetobacter xylinus (Brown 1886) Yamada et al. 1997,Gluconacetobacter hansenii (Gosselé et al. 1983) Yamada et al. 1997,Gluconacetobacter europaeus (Sievers et al. 1992) Yamada et al. 1997 (�‘Acetobacter polyoxogenes’ Entani et al. 1985), Gluconacetobacteroboediens (Sokollek et al. 1998) Yamada 2000, Gluconacetobacterintermedius (Boesch et al. 1998) Yamada 2000 and Gluconacetobacterentanii Schüller et al. 2000.

It is noted that the vinegar made from ‘sake lees’ or rice has been usedespecially for ‘sushi’ in Japan. In vinegar fermentation, there are twomethods: surface and submerged cultures. In the surface culture, a mixtureof heterogeneous microorganisms or a starter of ‘seed vinegar’ isgenerally employed. Strains identified as Acetobacter pasteurianus(Hansen 1879) Beijerinck & Folpmers 1916 are dominant organisms (1).Strains of G. xylinus are rather harmful in the surface vinegar

I-8

fermentation. The submerged culture is divided into two. One is the‘medium acid-producing’ submerged culture (5-10% acetic acidproduced), in which cellulose-less strains of G. xylinus play an importantrole. The other is the ‘high acid-producing’ submerged culture (10-20%acetic acid produced), from which strains classified and named ‘A.polyoxogenes’ were isolated (2).

Many new species and new combinations were recently described in thegenus Acetobacter (3,4). Accordingly, the vinegar-producing acetic acidbacteria mentioned above will be re-identified from the viewpoint of themicrobial systematics.

1) E. Entani and H. Masai, J. Brew. Soc. Jpn., 79, 746 (1984). 2) E.Entani, S. Ohmori, H. Masai and K. Suzuki, J. Gen. Appl. Microbiol., 31,475 (1985). 3) P. Lisdiyanti, H. Kawasaki, T. Seki, Y. Yamada, T.Uchimura and K. Komagata, J. Gen. Appl. Microbiol., 46, 147 & 47, 119(2000 & 2001). 4) L. Cleenwerck, K. Vandemeulebroecke, D. Janssensand J. Swings, Int. J. Syst. Evol. Microbiol., 52, 1551 (2002). *SeniorOverseas Volunteer of JICA; e-mail, yamada@biotec.or.th.

Introduction

The acetic acid bacteria that are accommodated in the familyAcetobacteraceae Gillis & De Ley 1980, the α-ProteobacteriaStackebrandt et al. 1988 are nowadays classified in the following sixgenera: Acetobacter Beijerinck 1898, Gluconobacter Asai 1935,Acidomonas Urakami et al. 1989, Gluconacetobacter Yamada et al. 1997,Asaia Yamada et al. 2000 and Kozakia Lisdiyanti et al. 2002 (1-3). Of thesix genera, the acetic acid bacteria that are utilized industrially for vinegarfermentation are distributed mainly in the genera Acetobacter andGluconacetobacter. Many new species and new combinations wererecently proposed in the genus Acetobacter (4-7). Accordingly, thevinegar-producing acetic acid bacteria will be re-identified from theviewpoint of the ‘new’ microbial systematics.

This paper describes industrial vinegar production in Japan and recentprogress in the classification of vinegar-producing acetic acid bacteria.

Vinegar fermentation

It is noted that the vinegar made from ‘sake lees’ or rice has been usedtraditionally for ‘sushi’ in Japan. For the industrial production of vinegar,there have been several methods. However, the methods are roughlydivided into two: the traditional surface culture and the modern submergedculture (8).

In the traditional surface culture, the mixture of heterogeneous, but notisolated, microorganisms or a starter of ‘seed vinegar’ is used industrially.Strains identified as Acetobacter pasteurianus (Hansen 1879) Beijerinck& Folpmers 1916 are dominant organisms (9). Strains (‘konnyaku kin’) ofGluconacetobacter xylinus (Brown 1886) Yamada et al. 1997, which formcellulosic polysaccharides, are rather harmful in the surface vinegarfermentation. The submerged culture is divided into two. One is the‘medium acid-producing’ submerged culture (5-10% acetic acidproduced), in which cellulose-less strains of G. xylinus play an importantrole. The other is the ‘high acid-producing’ submerged culture (10-20%acetic acid produced), from which strains classified and named‘Acetobacter polyoxogenes’ Entani et al. 1985 were isolated (10).

In the genus Gluconacetobacter (1), the vinegar-producing acetic acidbacteria are phylogenetically restricted only to sublineage B (1-3), whichis composed of G. xylinus, Gluconacetobacter hansenii (Gosselé et al.1983) Yamada et al. 1997, Gluconacetobacter europaeus (Sievers et al.1992) Yamada et al. 1997 (� ‘A. polyoxogenes’) (11), Gluconacetobacteroboediens (Sokollek et al. 1998) Yamada 2000 (4), Gluconacetobacterintermedius (Boesch et al. 1998) Yamada 2000 (12) andGluconacetobacter entanii Schüller et al. 2000 (13).

Classification of acetic acid bacteria assigned to the generaAcetobacter and Gluconacetobacter

The genus AcetobacterIn the Approved Lists of Bacterial Names, 1980 (14), three species werelisted in the specific-level classification of the genus Acetobacter:

Acetobacter aceti (Pasteur 1864) Beijerinck 1898 (type species), A.pasteurianus and Acetobacter peroxydans Visser’t Hooft 1925. In theformer two species, several subspecies were recognized: A. aceti subsp.aceti, A. aceti subsp. liquefaciens (Asai 1935) De Ley & Frateur 1974, A.aceti subsp. orleanensis (Henneberg 1906) De Ley & Frateur 1974, A.aceti subsp. xylinum (Brown 1886) De Ley & Frateur 1974, A.pasteurianus subsp. pasteurianus, A. pasteurianus subsp. ascendens(Henneberg 1898) De Ley & Frateur 1974, A. pasteurianus subsp.estunensis (Carr 1958) De Ley & Frateur 1974, A. pasteurianus subsp.lovaniensis (Frateur 1950) De Ley & Frateur 1974 and A. pasteurianussubsp. paradoxus (Frateur 1950) De Ley & Frateur 1974, The above-mentioned taxonomic system was derived from Bergey’s Manual ofDeterminative Bacteriology, 8th Edition, 1974 (15).

Gosselé et al. (16) examined in 1983 strains assigned to the genusAcetobacter for numerical analysis of phenotypic features and protein gelelectrophoresis. Their conclusion was that the above-mentionedsubspecies should be ruled out. The genus Acetobacter was divided intofour groups at the specific level: Acetobacter liquefaciens (Asai 1935)Gosselé et al. 1983 (phenon 2: new combination), Acetobacter hanseniiGosselé et al. 1983 (subphenon C: new species), A. aceti (subphenon D:type species) and A. pasteurianus (subphenon E). However, A.pasteurianus had an extremely wide range of DNA G+C contents, whichwere from 52.8 through 62.5 mol% with a range of 9.7 mol%. The 9.7-mol% range suggested especially a heterogeneous nature of this taxon.

Acetobacter xylinus (Brown 1886) Yamada 1983, a new combination wasproposed for strains characterized by ubiquinone-10 (Q-10) in contrast toubiquinone-9 (Q-9) of strains of A. aceti and A. pasteurianus (17). Thetype strain of this species was actually a member of A. pasteurianusemend. Gosselé et al. 1983 (16).

Sokollek et al. (4) described the following two new species: Acetobacterpomorum Sokollel et al. 1998 and Acetobacter oboediens Sokollek et al.1998. The latter species was later transferred to the genusGluconacetobacter on the basis of the 16S rRNA gene sequences and themajor quinone comprised of Q-10 (18).

Lisdiyanti et al. (5,6) examined strains assigned to the genus Acetobacteras well as isolates from Indonesian sources for DNA G+C contents andDNA-DNA hybridization. They recognized A. pasteurianus and A.peroxydans in addition to the type species, A. aceti. The proposed newspecies and new combinations were: Acetobacter indonesiensis Lisdiyantiet al. 2000, Acetobacter tropicalis Lisdiyanti et al. 2000, Acetobactersyzygii Lisdiyanti et al. 2001, Acetobacter cibinongensis Lisdiyanti et al.2001, Acetobacter orientalis Lisdiyanti et al. 2001, Acetobacterorleanensis (Henneberg 1906) Lisdiyanti et al. 2000, Acetobacterlovaniensis (Frateur 1950) Lisdiyanti et al. 2000 and Acetobacterestunensis (Carr 1958) Lisdiyanti et al. 2000.

Cleenwerck et al. (7) subsequently described additional new species on thebasis of DNA-DNA hybridization. The proposed new species were:Acetobacter cerevisiae Cleenwerck et al. 2002 and Acetobacter malorumCleenwerck et al. 2002.

The species mentioned above of the genus Acetobacter werephylogenetically divided into two sublineages, A and B (2,5-7). Theformer includes A. aceti (type species) characterized phenotypically by theproduction of 2-keto-D-gluconate from glucose, and the latter includes A.pasteurianus characterized by no production of 2-keto-D-gluconate (5,6).

The genus GluconacetobacterYamada and Kondo (19) divided the genus Acetobacter into twosubgenera on the basis of the difference in the ubiquinone system. Thetype subgenus Acetobacter was characterized by Q-9. In contrast, thesubgenus Gluconacetobacter Yamada & Kondo 1984 was characterizedby Q-10. To the latter subgenus, the two species were transferred:Acetobacter (Gluconacetobacter) liquefaciens and Acetobacter(Gluconacetobacter) xylinus. However, the subdivision to the twosubgenera was not recognized by Swings (20) along with the name of thegenus Acidomonas Urakami et al. 1989, which is comprised only ofmethanol-assimilating acetic acid bacteria.

The subgenus Gluconacetobacter was in 1997 elevated to the genericlevel on the basis of the partial 16S rRNA sequence analyses (1). The

genus Gluconacetobacter Yamada et al. 1997 was proposed with thefollowing new combinations: Gluconacetobacter liquefaciens (Gosselé etal. 1983) Yamada et al. 1997 (type species), Gluconacetobacterdiazotrophicus (Gillis et al. 1989) Yamada et al. 1997, G. xylinus, G.hansenii and G. europaeus (� ‘A. polyoxogenes’).

The species accommodated or transferred to the genus Gluconacetobacterwere: G. oboediens, G. intermedius, Gluconacetobacter sacchari Frankeet al. 1999 (21), G. entanii, Gluconacetobacter johannae Fuentes-Ramírezet al. 2001 (22) and Gluconacetobacter azotocaptans Fuentes-Ramírez etal. 2001 (22).

Navarro and Komagata (23) reported in 1999 that G. liquefaciens and G.xylinus are differentiated from each other molecular-biologically andphenotypically. In addition, G. hansenii had a quite heterogeneous naturein the phenotypic characteristics including the ubiquinone system (Q-10and Q-9), DNA base composition and DNA-DNA similarities (24).

The species mentioned above of the genus Gluconacetobacter werephylogenetically divided into two sublineages, A and B (1-3). The formerincludes G. liquefaciens (type species), and the latter includes G. xylinusand G. hansenii.

Classification of vinegar-producing acetic acid bacteria

It was quite recent that strains assigned to the genera Acetobacter andGluconacetobacter, especially to the genus Acetobacter, were investigatedin detail for the specific-level classification. Accordingly, the vinegar-producing acetic acid bacteria utilized industrially should be re-investigated and re-identified from the viewpoint of the ‘new’ microbialsystematics. However, there are a few reports (25,26).

Summary

The vinegar made from ‘sake lees’ or rice has been used traditionally for‘sushi’ in Japan. Strains identified as A. pasteurianus are dominantorganisms in the traditional surface culture. In the ‘medium acid-

producing’ submerged culture (5-10% acetic acid produced), cellulose-less strains of G. xylinus play an important role. In the ‘high acid-producing’ submerged culture (10-20% acetic acid produced), strainsclassified and named ‘A. polyoxogenes’ were isolated.

The recent progress in the classification of strains assigned to the generaAcetobacter and Gluconacetobacter, especially to the genus Acetobacter,proposed many new species and new combinations. Accordingly, thevinegar-producing acetic acid bacteria utilized industrially should be re-investigated and re-identified taxonomically.

References

1) Yamada, Y., Hoshino, K. and Ishikawa, T. (1997). The phylogenyof acetic acid bacteria based on the partial sequences of 16S ribosomalRNA: The elevation of subgenus Gluconoacetobacter to the generic level.Biosci. Biotechnol. Biochem., 61, 1244-1251.

2) Yamada, Y., Katsura, K., Kawasaki, H., Widyastuti, Y., Saono, S.,Seki, T., Uchimura, T. and Komagata, K. (2000). Asaia bogorensis gen.nov., sp. nov., an unusual acetic acid bacterium in the α–Proteobacteria.Int. J. Syst. Evol. Microbiol., 50, 823-829.

3) Lisdiyanti, P., Kawasaki, H., Widyastuti, Y., Saono, S., Seki, T.,Yamada, Y., Uchimura, T. and Komagata, K. (2002). Kozakia baliensisgen. nov., sp. nov., a novel acetic acid bacterium in the α–Proteobacteria.Int. J. Syst. Evol. Microbiol., 52, 813-818.

4) Sokollek, S.J., Hertel, C. and Hammes, W.P. (1998). Description ofAcetobacter oboediens sp. nov. and Acetobacter pomorum sp. nov., twonew species isolated from industrial vinegar fermentations. Int. J. Syst.Bacteriol., 48, 935-940.

5) Lisdiyanti, P., Kawasaki, H., Seki, T., Yamada, Y., Uchimura, T.and Komagata, K. (2000). Systematic study of the genus Acetobacter withdescriptions of Acetobacter indonesiensis sp. nov., Acetobacter tropicalissp. nov., Acetobacter orleanensis (Henneberg 1906) comb. nov.,Acetobacter lovaniensis (Frateur 1950) comb. nov. and Acetobacterestunensis (Carr 1958) comb. nov. J. Gen Appl. Microbiol., 46, 147-165.

6) Lisdiyanti, P., Kawasaki, H., Seki, T., Yamada, Y., Uchimura, T.and Komagata, K. (2001). Identification of Acetobacter strains isolatedfrom Indonesian sources and proposals of Acetobacter syzygii sp. nov.,Acetobacter cibinongensis sp. nov. and Acetobacter orientalis sp. nov. J.Gen Appl. Microbiol., 47, 119-131.

7) Cleenwerck, I., Vandemeulebroecke, K, Janssens, D. and Swings, J.(2002). Re-examination of the genus Acetobacter, with descriptions ofAcetobacter cerevisiae sp. nov. and Acetobacter malorum sp. nov. Int. J.Syst. Evol. Microbiol., 52, 1551-1558.

8) Entani, E. (2001). Shokusu (Vinegar). In Hakko Handbook(Fermentation Handbook), ed. by Tochikura, T., Yamada, H., Beppu, T.and Sohda, K., Kyoritsu Shuppan, Tokyo, pp. 599-604 (in Japanese).

9) Entani, E. and Masai, H. (1984). Shokusu jozo ni okeru yuhryosakusankin no bunri to seishitsu (Isolation and characterization ofexcellent acetic acid bacteria from fermenting vinegar). NipponJozokyokai Zasshi (J. Brew. Soc. Jpn.), 79, 746-750 (in Japanese).

10) Entani, E., Ohmori, S., Masai, H. and Suzuki, K. (1985).Acetobacter polyoxogenes sp. nov., a new species of an acetic acidbacterium useful for producing vinegar with high acidity. J. Gen. Appl.Microbiol., 31, 475-490.

11) Sievers, M., Sellmar, S. and Teuber, M. (1992). Acetobactereuropaeus sp. nov., a main component of industrial vinegar fermenters incentral Europe. Syst. Appl. Microbiol., 15, 386-392.

12) Boesch, C., Trček, J., Sievers, M. and Teuber, M. (1998).Acetobacter intermedius sp. nov. Syst. Appl. Microbiol., 21, 220-229.

13) Schüller, G., Hertel, C. and Hammes, W.P. (2000).Gluconacetobacter entanii sp. nov., isolated from submerged high-acidindustrial vinegar fermentations. Int. J. Syst. Evol. Microbiol., 50, 2013-2020.

14) Skerman, V.B.D., McGowan, V. and Sneath, P.H.A. (1980).Approved lists of bacterial names. Int. J. Syst. Bacteriol., 30, 225-420.

15) De Ley, J. and Frateur, J. (1974). Genus Acetobacter Beijerinck1898. In Bergey’s Manual of Determinative Bacteriology, 8th Edition, ed.by Buchanan, R.E. and Gibbons, N.E., Williams & Wilkins, Baltimore, pp.276-278.

16) Gosselé, F., Swings, J., Kersters, K., Pauwels, P. and De Ley, J.(1983). Numerical analysis of phenotypic features and protein gelelectrophoregrams of a wide variety of Acetobacter strains. Proposal forthe improvement of the taxonomy of the genus Acetobacter Beijerinck1898, 215. Syst. Appl. Microbiol., 4, 338-368.

17) Yamada, Y. (1983). Acetobacter xylinus sp. nov., nom. rev., for thecellulose-forming and cellulose-less, acetate-oxidizing acetic acid bacteriawith the Q-10 system. J. Gen. Appl. Microbiol., 29, 417-420.

18) Yamada, Y. (2000). Transfer of Acetobacter oboediens Sokollek etal. 1998 and Acetobacter intermedius Boesch et al. 1998 to the genusGluconacetobacter as Gluconacetobacter oboediens comb. nov. andGluconacetobacter intermedius comb. nov. Int. J. Syst. Evol. Microbiol.,50, 2225-2227.

19) Yamada, Y. and Kondo, K. (1984). Gluconoacetobacter, a newsubgenus comprising the acetate-oxidizing acetic acid bacteria withubiquinone-10 in the genus Acetobacter. J. Gen. Appl. Microbiol., 30,297-303.

20) Swings, J. (1992). The genera Acetobacter and Gluconobacter. InThe Prokaryotes, 2nd Edition, Vol. 3, ed. by Balows, A., Trüper, H.G.,Dworkin, M. and Schleifer, K.-H., Springer-Verlag, New York, pp. 2268-2286.

21) Franke, I.H., Fegan, M., Hayward, C., Leonard, G., Stackebrandt,E. and Sly, L.I. (1999). Description of Gluconacetobacter sacchari sp.nov., a new species of acetic acid bacterium isolated from the leaf sheathof sugar cane and from the pink sugar-cane mealy bug. Int. J. Syst.Bacteriol., 49, 1681-1693.

22) Fuentes-Ramírez, L.E., Bustillos-Cristales, R., Tapia-Hernádez, A.,Jiménes-Salgado, T., Wang. E.T., Martínez-Romero, E. and Caballero-Mellado, J. (2001). Novel nitrogen-fixing acetic acid bacteria,

Gluconacetobacter johannae sp. nov. and Gluconacetobacterazotocaptans sp. nov., associated with coffee plants. Int. J. Syst. Evol.Microbiol., 51, 1305-1314.

23) Navarro, R.R. and Komagata, K. (1999). Differentiation ofGluconacetobacter liquefaciens and Gluconacetobacter xylinus on thebasis of DNA base composition, DNA relatedness and oxidation productsfrom glucose. J. Gen. Appl. Microbiol., 45, 7-15.

24) Navarro, R.R., Uchimura, T. and Komagata, K. (1999). Taxonomicheterogeneity of strains comprising Gluconacetobacter hansenii. J. Gen.Appl. Microbiol., 45, 295-300.

25) Trček, J. and Raspor, P. (1999). Molecular characterization ofacetic acid bacteria isolated from spirit vinegar. Food Technol.Biotechnol., 37, 113-116.

26) Du Toit, W.J. and Lambrechts, M.G. (2002). The enumeration andidentification of acetic acid bacteria from South African red winefermentations. Int. J. Food Microbiol., 74, 57-64.

*Senior Overseas Volunteer of JICA; e-mail,yamada@biotec.or.th/yamada-yuzo@mub.biglobe.ne.jp