INTERNATIONALJOURNAL OF SYSTEMATICBACTERIOLOGY, Oct. 1991, p. 588-589 Vol. 41, No. 4 0020-7713/91/040588-02$02.OO/O Copyright 0 1991, International Union of Microbiological Societies

Strain GP6 Is Proposed as the Neotype Strain of soehngenii*-VP pro synon. Methanothrix concilii-VP and concilii’ 9VP Request for an Opinion DAVID R. BOONE Departments of Environmental Science and Engineering and Chemical and Biological Sciences, Oregon Graduate Institute, 19600 N.W. von Neumann Dv.,Beaverton, Oregon 97006-1999

No axenic cultures of OpfikonT (T = type strain), exist, nor is any extant contaminated culture known to be derived from strain Opfikon‘r. I propose strain GP6 as the neotype strain of this taxon. Strain GP6 is the type strain of and Methanothrix concilii, but both of these names are widely considered to be subjective synonyms of Methanothrix soehngenii. ______Three names for the aceticlastic, mesophilic, vancomycin per liter) to inhibit growth of Contaminants. sheathed, rod-shaped methanogens (Methanothrix soehnge- However, when cultures of strain OpfikonT were transferred nii, Methanothrix concilii, and Methanosaeta concilii) are four times in antibiotic-free medium and then inoculated into currently in use, yet these names are widely regarded as fluid thioglycolate medium, this method still revealed no synonymous. The correct name is at present controversial contaminants (9), implying that the culture was pure at the because of uncertainty concerning whether the description time. In a recent poll of the members of the Subcommittee of Methanothrix soehngenii was based upon a pure culture for of Methanogens, one-half of the respondents (strain OpfikonT [T = type strain]). If strain OpfikonT was (9 of 18) agreed that we should assume that the description of never axenic, the description of Methanothrix soehngenii Methanothrix soehngenii was based on a pure culture. has not been validly published (Rule 31a [3]). Patel, who also used the isolation technique of dilution in Rod-shaped aceticlastic methanogens have been known liquid medium, obtained strain GP6, a rod-shaped, aceticlas- since 1906, when they were first described by Sohngen (7). tic methanogen which he designated the type strain of Later, Barker (1) studied enrichment cultures of these or- Methanothrix concilii (5). This strain has been deposited in ganisms, calling them “Methanobacterium soehngenii.” several culture collections (strain GP6T = DSM 3671T = These methanogens eluded isolation for many years because OCM 69T = NRC 2989T = ATCC 35969T). Methanothrix they grow slowly (with doubling times of several days), they concilii was differentiated from Methanothrix soehngenii on are strict anaerobes, and in agar medium they grow poorly or the basis of several physiological characteristics and the not at all. Zehnder and coworkers developed enrichment G+C content of its DNA (3,but later studies (8) indicated cultures with very low numbers of contaminants by includ- that the two type strains (GP6 and DSM 2139) have DNAs ing vancomycin (0.1 g/liter) in the medium along with with nearly identical G+C contents and exhibit about 100% as the sole carbon and energy source. Serial dilutions of interstrain DNA hybridization; Touzel et al. (8) considered these cultures yielded a liquid culture with no apparent Methanothrix concilii to be a subjective synonym of Meth- contaminants, which was named strain Opfikon (10) and anothrix soehngenii. Hybridization analysis allows checking later designated the type strain of Methanothrix soehngenii even impure cultures (in which the contaminant is (2). Strain OpfikonT was sent to the Deutsche Sammlung von suppressed) against pure cultures, such as strain GP6T, and Microorganismen for deposit, but the culture was not viable this method demonstrated that the principal microbe in (9). A pure, viable culture of strain OpfikonT was apparently strain DSM 2139T cultures (considered to be strain never deposited in a culture collection, and no pure culture OpfikonT) is very similar to strain GP6T. exists today. A Methanothrix culture from Zehnder’s labo- Patel and Sprott later suggested that the name Methano- ratory (9) was later sent to a third party who, thinking it to be thrix soehngenii is invalid because the description was based strain OpfikonT, deposited it in the Deutsche Sammlung von on an impure culture (strain OpfikonT) and proposed the new Mikroorganismen as strain OpfikonT (= DSM 2139T). This Methnnosaeta for the aceticlastic methanogenic rods culture was found to be contaminated (6). (6). The conclusion of these authors that strain OpfikonT was Whether the actual strain OpfikonT was ever in fact pure is never pure was based on indirect evidence. No demonstra- a matter of some controversy. Cultures were sent to a bly axenic, viable cultures of strain OpfikonT appear to have number of microbiologists and rigorously examined for ever been sent to other microbiologists or to any culture contaminants, but none was found to be axenic. However, collection. However, deposition in a culture collection is the strain may have become contaminated after Zehnder et recommended but is not an absolute requirement (Rule 18a al. completed the characterization on which the species and Recommendation 30a [3]). In the absence of direct description was based. Strain OpfikonT was described as a evidence that the culture was never pure, I believe that the pure culture (2, lo), but the methods used for testing culture published report of its purity should be accepted. In that purity are unlikely to have revealed contaminants. The case, Methanosaeta concilii is a junior objective synonym of cultures to be checked were inoculated into fluid thioglyco- Methanothrix concilii. Furthermore, the preponderance of late medium and incubated, but enough antibiotic might have evidence suggests that these names are subjective junior been transferred with the inoculum (the culture had 0.1 g of synonyms of Methanothrix soehngenii. This conclusion was

588 VOL.41, 1991 REQUEST FOR AN OPINION 589 also supported by the results of the poll of the Subcommittee ACKNOWLEDGMENTS for Taxonomy of Methanogens (15 of 16 respondents con- For helpful advice and suggestions, I thank G. B. Patel, A. J. B. sidered these names to be synonymous on the basis of the Zehnder, S. Zinder, Y. Kamagata, J. T. Staley, R. G. E. Murray, assumption that the description of Methanothrix soehngenii and the Subcommittee for Taxonomy of Methanogens (A. Antono- was based on a pure culture). poulos, D. Archer, B. Biavati, E. Conway de Macario, H.-C. The existence of even a contaminated culture of the Dubourguier, J. G. Ferry, H. Hippe, W. J. Jones, D. P. Kelly, Y. original strain OpfikonT is in doubt. Even if such a culture Koga, T. A. Langworthy, R. A. Mah, T. L. Miller, D. Searcy, E. could be identified, it is unlikely that it would be repurified Stackebrandt, K. 0. Stetter, C. van der Drift, W. B. Whitman, J. S. in the near future. Few workers have been successful at Wiegel, R. Wolfe, Y. Zhao, and T. N. Zhilina). such isolations, and Zehnder has indicated that he does not REFERENCES intend to repurify it (9). Therefore, I propose strain GP6 as 1. Barker, H. A. 1936. Studies upon the -producing bac- the neotype strain of Methanothrix soehngunii. The mem- teria. Arch. Mikrobiol. 7:420438. bers of the Subcommittee for Taxonomy of Methanogens 2. Huser, B. A., K. Wuhrmann, and A. J. B. Zehnder. 1982. expressed the opinion (13 of 15 respondents) that strain GP6 Methanothrix soehngenii gen. nov., sp. nov., a new ace- is appropriate as a neotype strain of Methanothrix soehn- totrophic non-hydrogen-oxidizing methane bacterium. Arch. genii (if Methanothrix soehngenii has been validly pub- Microbiol. 132:l-9. lished). 3. Lapage, S. P., P. H. A. Sneath, E. F. Lessel, V. B. D. Skerman, The two names proposed for thermophilic species of H. P. R. Seeliger, and W. A. Clark (ed.). 1975. International code of nomenclature of bacteria. 1975 Revision. American aceticlastic rods, Methanothrix thermoacetophila (4) and Society for Microbiology, Washington, D.C. Methanosaeta thermoacetophila (6), are each based on a 4. Nozhevnikova, A. N., and V. I. Chudina. 1985. Morphology of description of an impure culture. Patel and Sprott (6) indi- the thermophilic acetate bacterium Methanothrix thermoaceto- cated that an axenic methanogenic culture was obtained phila sp. nov. Microbiology (Engl. Transl. Mikrobiologiya) from the originally described but impure type culture. How- 53:618-624. ever, their description of the species is limited to a table of 5. Patel, G. B. 1984. Characterization and nutritional properties of data that were taken from the original description of the Methanothri-r concilii sp. nov., a mesophilic, aceticlastic meth- impure culture. Thus, this description was also based on an anogen. Can. J. Microbiol. 30:1383-1396. impure culture, regardless of the purported purity of the 6. Patel, G. B., and G. D. Sprott. 1990. Methanosaeta concilii gen nov., sp. nov. ("Methanothrix concilii") and Methanosaeta current culture of Patel and Sprott. The morphology of the thermoacrtophila nom. rev., comb. nov. Int. 1. Syst. Bacteriol. type strain is sufficiently distinctive that it may be distin- 40: 79-82. guishable as a member of the genus Methanothrix (or 7. Sohngen, N. L. 1906. Het ontstaan en verdwijnen van waterstof Methanosaeta), but it cannot be distinguished from Metha- en methaan onder den invloed van het organische leven. Ph. D. nothrix soehngenii except by a physiological characteristic thesis, University of Delft, Delft, The Netherlands. (namely, growth temperature). Hence, this strain cannot be 8. Touzel, J. P., G. Premier, J. L. Roustan, I. Thomas, H. C. named on the basis of morphology, especially with a specific Dubourguier, and G. Albagnac. 1988. Description of a new epithet which implies a physiological characteristic which it strain of Methanothrix soehngenii and rejection of Methano- is thrix concilii as a synonym of Methanothrix soehngenii. Int. J. reported to possess. Furthermore, since another strain of Syst. Bacteriol. 38:30-36. this species has apparently been isolated in axenic culture, it 9. Zehnder, A. J. B. (Wageningen Agricultural University, Wa- seems inappropriate to base the species description on an geningen, The Netherlands). 1991. Personal communication. impure culture (Rule 31a). Therefore, Methanothrix ther- 10. Zehnder, A. J. B., B. A. Huser, T. D. Brock, and K. Wuhrmann. moacetophila and Methanosaeta thermoacetophila are not 1980. Characterization of an acetate-decarboxylating, non-hydro- validly published names (Rule 31a). gen-oxidizing methane bacterium. Arch. Microbiol. 124:l-11.