University of Groningen Bacillus methanolicus sp. nov., a New Species of Thermotolerant, Methanol-Utilizing, Endospore-Forming Bacteria Arfman, Nico; Dijkhuizen, Lubbert; Kirchhof, Gudrun; Ludwig, Wolfgang; Schleifer, Karl-Heinz; Bulygina, Eugenia S.; Chumakov, Konstantin M.; Govorukhina, Natalya I.; Trotsenko, Yuri A.; White, Duncan Published in: International Journal of Systematic Bacteriology DOI: 10.1099/00207713-42-3-439 IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record Publication date: 1992 Link to publication in University of Groningen/UMCG research database Citation for published version (APA): Arfman, N., Dijkhuizen, L., Kirchhof, G., Ludwig, W., Schleifer, K-H., Bulygina, E. S., Chumakov, K. M., Govorukhina, N. I., Trotsenko, Y. A., White, D., & Sharp, R. J. (1992). Bacillus methanolicus sp. nov., a New Species of Thermotolerant, Methanol-Utilizing, Endospore-Forming Bacteria. International Journal of Systematic Bacteriology, 42(3), 439-445. https://doi.org/10.1099/00207713-42-3-439 Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license. More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne- amendment. Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. INTERNATIONAL JOURNAL OF SYSTEMATICBACTERIOLDGY, July 1992, p. 439445 Vol. 42, No. 3 0020-7713/92/030439-07$02.00/0 Copyright 0 1992, International Union of Microbiological Societies Bacillus methanolicus sp. nov., a New Species of Thermotolerant, Methanol-Utilizing, Endospore-Forming Bacteria NICO ARFMAN,l LUBBERT DIJKHUIZEN,'* GUDRUN KIRCHHOF,2 WOLFGANG LUDWIG,2 KARL-HEINZ SCHLEIFER,2 EUGENIA S. BULYGINA,3 KONSTANTIN M. CHUMAKOV,3 NATALYA I. GOVORUKHINA,4 YURI A. TROTSENK0,4 DUNCAN WHITE,^ AND RICHARD J. SHARP^ Department of Microbiology, Universiq of Groningen, NL-9751 NN Haren, The NetherlandsI; Lehrstuhl fur Mikrobiologie, Technische Universitat Miinchen, 0-8000 Munich, Germany2;Institute of Microbiology, Academy of Sciences, 11 7811 Moscow, and Institute of Biochemistry and Physiology of Microorganisms, Puschino, Moscow Region, Russia; and Public Health Laboratory Sewice Centre for Applied Microbiolo and Research Division of Biotechnology, Porton Down, Salisbury, Wiltshire SP4 OJG, United KingdomBy The generic position of 14 strains of gram-positive bacteria able to use methanol as a growth substrate was determined. All are obligately aerobic, thermotolerant organisms that are able to grow at temperatures of 35 to 60°C. Nine of the strains produce oval spores at a subterminal-to-central position in slightly swollen rod-shaped cells. DNA-DNA hybridization studies, 5s rRNA sequence analysis, and physiological character- istics revealed that all 14 strains cluster as a well-defined group and form a distinct new genospecies. Analysis of the 16s and 5s rRNA sequences indicated that this new species is distinct from BuciUus brevis but closely related to B.Jinnus and B. uzotoformuns. The name proposed for this new species is B. methunolicus. The type strain, PB1, has been deposited in the National Collection of Industrial and Marine Bacteria as NCIMB 13113. Bacteria able to grow on methanol at elevated tempera- genus. In the present investigation, 14 organisms, including tures are of technological interest for single-cell protein most of Al-Awadhi's isolates, were further characterized, production, solvent degradation in aerobic thermophilic and a classification of these strains based on various prop- biotreatment processes, and fermentative production of erties, including DNA-DNA hybridization, is proposed. amino acids (1,25,42). Following an earlier report on growth of a mixed culture with spore-forming bacteria on methanol (45), a claim appeared in the patent literature (31) concerning MATERIALS AND METHODS Bacillus strains that grow on methanol with optimum growth temperatures of ca. 55°C and maximum growth temperatures of ca. 65°C. In recent years, several other groups have Test strains and cultivation conditions. The thermotolerant successfully employed continuous culture techniques for methanol-utilizing Bacillus strains PB1 (NCIMB 13113), C1 isolation of pure cultures of Bacillus strains that grow rapidly (NCIMB 13114), AR2, TS1, TS2, and TS4, isolated by on methanol at 55°C (5, 15, 24, 29, 42). Metabolism of Dijkhuizen et al. (24); 4(55) (NCIMB 12523), S1 (NCIMB methanol, ethanol, and glucose (7, 9, 23, 47) and environ- 12524), WM5.2 (NCIMB 12525), TFB (NCIMB 12526), mental control of metabolic fluxes (2-4,14,15,30) have been WM5.1 (NCIMB 12527), S2 (NCIMB 12528), and JSA studied in detail in these organisms, grown in batch and (NCIMB 12529), isolated by Al-Awadhi et al. (5); and 40M, continuous cultures under a variety of nutrient limitations isolated by Govorukhina and Trotsenko (29), were the and steady-state as well as transient-state conditions. The subject of chemotaxonomic, molecular systematic, and phe- methylotrophic Bacillus strains display a strong resistance to notypic tests. The mesophilic trimethylamine-utilizing Bacil- high methanol concentrations, and the molar growth yields lus strain S2A1 (19) was included in the 5s rRNA sequence on methanol at the optimum growth temperatures in metha- analysis. The nonmethylotrophs B. firmus DSM 12 and B. nol-limited chemostats are among the highest reported for brevis DSM 30 were used as reference strains in some of the methylotrophic bacteria (6, 24). Enzyme analysis revealed physiological tests. All strains were stored without supple- that all isolates employ a novel NAD-dependent methanol ments as frozen stocks at -80°C and routinely grown in dehydrogenase for methanol oxidation and the ribulose Tryptone soya broth (TSB; Oxoid CM131; 30 g/liter) ad- monophosphate pathway for formaldehyde assimilation (7- justed to pH 7.5. For plates, TSB was solidified with 1.5% (wthol) agar (TSBA). Growth of the methylotrophic Bacillus 9). On the basis of a number of phenotypic tests, Al-Awadhi strains in batch cultures and carbon-, oxygen- or nitrogen- et al. (5) concluded that seven of their isolates were Bacillus limited continuous cultures has been described previously brevis strains. A further isolate was a sheathed, filamentous, (7, 9, 14). DNA, 16s rRNA, 5s rRNA, cell wall fractions, gram-positive, endospore-forming, obligately aerobic bacte- fatty acids, and lipids were isolated from cells grown in batch rium that could not be allocated to any previously described cultures in trimethylamine (25 mM; strain S2A1 only) or methanol (100 mM; all other strains) mineral medium sup- plied with vitamins (24). Morphology. Colony morphology was examined in iso- * Corresponding author. lated colonies grown on TSBA for 2 days at 55°C. Cellular 439 440 ARFMAN ET AL. INT. J. SYST.BACTERIOL. 1 UUUUUGGAGA GWUGAUCCU GGCUCAGGAC GAACGCUGGC GGCGUGCCUA AUApUacaa morphology was examined in gram-stained smears of these 61 gucaaacqGA CUGAUGGGAG CWGCUCCCU GAAGUCAGCG GCGGACGGGU GAGUAACACG cultures. Cell dimensions were measured by a calibrated 121 UGGGCAACCU GCCUGUAAGA CCGGGAUAAC UUCGGGAAAC CGGAGCUAAU ACCGGAUAAU eyepiece graticule. Cell diameters were recorded as being 181 CCUCUUUCCC GCAUGGGAAA GAGCUGAAAG AUGGCUUCGG CUAUCACUUA CAGAUGGGCC either less than 1 pm (code = 0) or greater than 1 pm (code 241 CGCGGCGCAU UAGCUAGWG GUGAGGUAAC GGCUCACCAA GGCGACGAUG CGUAGCCGAC = 2). Cell lengths were recorded as either less than 1 pm (code = 0), between 4 and 6 pm (code = l),or greater than 301 CUGAGAGGGU GAUCGGCCAC ACUGGGACUG AGACACGGCC CAGACUCCUA C- 6 pm (code = 2). Spore morphology was examined in 361 UUAGGGAA UCUUCCGCAA UGGACGAAAG UCUGACGGAG CAACGCCGCG UGAGCGAAGA cultures grown on mannitol mineral medium plates (1% 421 AGGCCUUCGG GUCGUAAAGC UCUGUUGUCA GGGAAGAACA AGUACCGUUC GAAUAGGGCC [wt/vol] mannitol, 1.5% [wt/vol] agar) for 5 days. The 481 GUACUUUGAC GGUACCUAAC CAGAAAGCCA CGGCUAACUA CGUGCCAGP GCCGCGGUAA mineral medium used was that described by Dijkhuizen et al. 541 XGUAGGUG GCAAGCGWG UCCGGAAUUA UUGGGCGUAA AGCGCGCGCA GGCGGUUCCU (24). 601 UAAGUCUGAU GUGAAAGCCC ACGGCUCAAC CGUGGAGGGU CAUUGGAAAC UGGGGAACW Physiological tests. Tests with the thermotolerant methylo- 661 GAGUGCAGAA GAGGAGAGCG GAAUUCCACG UGUAGCSUG AllAUGCGUAG AGAUGUGGAG trophic Bacillus strains were performed at 55°C. Tests with 721 GAACACCAGU GGCGAAGGCG GCUCUCUGGU CUGUAACUGA CGCUGAGGCG CGAAAGCGUG reference strains B. firmus and B. brevis were performed at 781 GGGAGCAAAC AGGAWAGAU ACCCUGGUAG UCCACGCCGU AAACGAUGAG UGCUAAGUGU 37°C. All tests were inoculated with overnight peptone water broth cultures derived from overnight plate cultures. Acid 841 UAGAGGGWU CCGCCCUUUA GUGCUGCAGC UAACGCAUUA AGCACUCCGC CUGGGGAGUA production from sugars and sugar alcohols and hippurate 901 CGGCCGCAAG GCUGAAACUC AAAGGAAUUG ACGGGGGCCC GCACAAGCGG UGQAGCAUGU hydrolysis were detected by using the media and methods of 961 GGUUUAAUUC GAAGCAACGC GAAGAACCUU ACCAGGUCUU GACAUCCUCU GACAAUCCUG