J. Gen. Appl. Microbiol., 18, 295-305 (1972) NEW YEASTS CAPABLE OF ASSIMILATING METHANOL* TOSHIKAZU OKI, KAGEAKI KOUNO, ATSUO KITAI, ANDASAICHIRO OZAKI Central Research Laboratories of Sanraku- Ocean Co., Ltd., Fujisawa, Japan (Received May 10, 1972) Twenty strains of methanol strongly assimilating yeasts were isolated from rotten tomato and a flower of azalea through investigations on the single- cell protein production and on the microbial utilization of Cl compound. Taxonomic studies indicated that these yeasts were limited to certain species of Candida and Torulopsis, including two new species; C. methanolica OKi et KouNo sp. nov. and T. methanolovescens OKi et KouNo sp. nov. One hundred and ninety-one strains of yeasts obtained from type culture collections did not exhibit methanol assimilability at all. The possibility of producing a single-cell protein from methanol by micro- organisms was suggested by the extensive studies concerning methane- or methanol-assimilating bacteria, Pseudomonas sp. PRL-W4 (1), Pseudomonas methanica (2, 3, 4), Methanomonas methanooxidans (5, 6), Pseudomonas AM 1(7 ), Pseudomonas sp. M27 (8), and Vibrio extorquens (9). Moreover, it is of considerable interest that OGATA et al. first reported the assimila- tion of methanol by yeast, Kloeckera sp. No. 2201 (10, 11, 12). More recently, ASTHANA et al. (13) isolated one species of yeast capable of uti- lizing methanol as a primary carbon source and identified it tentatively as Torulopsis glabrata. However, its taxonomical characteristics have not been reported yet. In the course of investigations on yeast production and microbial utiliza- tion of Ci compounds, two new species of yeasts assimilating methanol as a carbon and energy source were isolated from natural sources. In this paper the results of screening tests on methanol utilizers from a large variety of yeasts which were freshly isolated from various sources or obtained from several type culture collections, and taxonomical studies of new isolates are described. * This paper was presented at the Annual Meeting of the Agricultural Chemical Society of Japan held on April 4, 1969, in Fukuoka, Japan. 295 296 OKI, KouNo, KITAI, and OzAKI VOL. 18 Table 1. Composition of methanol medium. MATERIALS AND METHODS Microorganisms. Yeasts, 192 strains belonging to 13 genera, were used in this study. A part of them were obtained from culture collections of the Institute of Applied Microbiology, University of Tokyo (IAM), and of the Institute for Fermentation, Osaka (IFO). The other has been maintained in our laboratory. In additions, 20 strains of methanol-assimilating yeasts were isolated from natural sources by a methanol enrichment culture. Medium. Various media shown in Table 1 were used for the isolation, purification, and the assimilation test for methanol. Isolation of methanol-assimilating yeasts by a methanol enrichment culture. All methanol-assimilating yeasts were isolated by a methanol enrichment culture using media A, B, C, and D. Small amounts of soil, sewage, compost, humus, vegetables, and flowers were added to each medium of pH 3, 4, or 5 in Monod-shaking tubes, and incubated with reciprocal shaking for 1 week at 28°. After several subcultures, methanol-assimilating yeasts were plated on a methanol agar plate (medium D), and then transplanted to a nutrient- methanol broth (medium E) to be incubated for 48 hr at 28°. Yeasts growing in the latter medium were reisolated in "pour plates" of medium D and later placed on slants of the same medium. Further purification of the yeast was carried out by two repetitions of shaking culture and plating using media D and E alternatively. Screening test for methanol-assimilating yeasts. Methanol utilizers were preliminarily screened by the growth test on methanol-containing agar plates 1972 Methanol-assimilating Yeasts 297 (medium A, B, C, D, or E) for 1 week at 28°. The strains showing positive or doubtful results were retested by using a liquid medium. The cells grown on methanol agar slant were cultivated in a Monod-shaking tube containing 10 ml of medium A, B, D, or E with reciprocal shaking at 28°. After 36 to 72 hr, the ability of assimilation was detected by an increase in turbidity of the culture broth and the consumption of methanol in the medium determined by gas chromatography. Assimilation of methanol by stock cultures and type strains was confirmed with the growth on a methanol agar plate and in the liquid medium, and by the determination of methanol consumed, re- spectively. Identification of yeasts. The experimental methods were mainly those described by LODDER and KREGER-VAN RIJ (14 ), and results were discuss ed according to the system of LODDER (15). In some cases, 1 or 2% of methanol was added to the medium for identification, and detailed comparison with authentic cultures was carried out. Vitamin and amino acid require- ments were determined by using a minimum medium composed of methanol 20 ml, (NH4)2SO4 3 g, KH2PO4 2 g, MgSO4.7H2O 0.5 g, Mn2+ 2 mg, Fe2+ 2 mg, Bromocresol Green 2 mg, and distilled water 1,000 ml ; pH 5.0. RESULTS AND DISCUSSION Methanol assimilation by yeasts was carefully detected by three different methods ; growth on the methanol-containing agar plate, an increase of turbidity in the methanol-containing liquid medium, and determination of methanol consumption. The results of screening test for methanol-assimilat- ing yeasts are shown in Table 2. None of the 192 strains of type cultures and stock cultures preserved in our laboratory showed any the growth on a methanol medium. This is in good agreement with the results of OGATA et at. (10) who showed the in- ability of methanol assimilation by 38 strains of type culture of yeasts. All the strains assimilating methanol were newly isolated from natural sources. We isolated 191 colonies of methanol-assimilating yeasts from two samples, rotten tomato and a rotten flower of azalea, out of 51 samples of soil, humus, plants, sewage, and water, and selected 31 colonies which showed abundunt growth in methanol medium. These colonies are reproduced by multilateral budding, and were confirmed to be asporogenous yeast, not belonging to Sporobolomycetaceae, since they do not form ascospores, teliospores, bal- listospores, or arthrospores. The most abundunt growth in a methanol medi- um was shown by 20 strains of yeast, 14 of which were globose, ovoid, cylindrical, or elongate in shape (Fig. 1A), and formed well-developed pseu- domycelia, which sometimes developed into blastospores, as shown in Fig. lB and C. They fermented glucose well, did not assimilate inositol, and their streak cultures were not pigmented. Therefore, these 14 strains should belong to the genus Candida. 298 OKI, KouNo, KITAI, and OzAKI VOL. 18 Table 2. Results of screening of methanol-assimilating yeasts. On the other hand, 6 strains of yeast, globose or ovoid, do not form true mycelia and pseudomycelia, and have neither the assimilability of inositol, fermentation, nor pigmentation. These isolates, therefore, might be related to the genus Toyulopsis. These powerful methanol-utilizers were restricted to certain species of Candida and Toyulopsis isolated from rotten tomato and a rotten flower of azalea, and they were identified and named Candida met hanolica OKI et KouNo sp. nov. and Toyulopsis methanolovescens OKI et KouNo sp. nov. by the authors. Detailed taxonomical characteristics are as follows : 1. Candida methanolica OKI et KouNo sp. nov. Strains : No. 7 (FERM No. 1108), 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20. Fig. 1. Cells and pseudomycelia of Candida methanolica No. 7. A : Cells grown on MY agar (malt extract 3 g, yeast extract 3 g, peptone 5 g, glucose 10 g, agar 20 g, and distilled water 1,000 ml ; pH 5'6) (15) for 3 days at 25°. B : Pseudomycelia, slide culture on PDA agar (potato extract 200 g, dextrose 20 g, agar 15 g, and distilled water 1,000 ml; pH 5.6, "Nissan") (15) for 4 days at 25°. C : Blastospores, slide culture on PDA agar for 7 days at 25°. 1972 Methanol-assimilating Yeasts 299 300 OKI, K0UNO, KITAI, and OzAKI VOL. 18 Cellulae in extracto malti, ovatae, longovatae aut longae, raro aequales formae, (1-5) x (2-20) p, volgo catenatae. Anulus nut pellicula formatur. Cultura in agare (post sex dies, 28°) eborina aut subbrunnea, rugosa, margine erosa. Cultura in agare methanolico est eborine, non perlucida, non lucida, umbilicaris et aspra in superficie. Pseudomycelium formatur. D-Glucosum fermentatur at non D-galactosum, saccharosum, melibiosum, lactosum, in- ulinum, dextrinum, inositolum, maltosum, rafnosum nec a-methyl-D-glu- cosidum. In medio minerali D-glucosum, D-ribosum, L-rhamnosum, ethanolum, glycerolum, erythritolum, trehalosum, inulinum, amylum solbile, D-xylosum, L-arabinosum, D-glucitolum, D-mannitolum, adonitolum, salicinum, acidum DL- lacticum, et acidum succinicum assimilantur at non D-galactosum, L-sorbosum, saccharosum, maltosum, cellobiosum, lactosum, melibiosum, rafrinosum, mele- zitosum, D-arabinosum, dulcitolum, a-methyl-D-glucosidum, potassium gluco- natum, calcium 2-ketogluconatum, acidum citricum nec inositolum. Et etiam methanolum, fons carbonarius, utitur. Nitras kalicus assimilatur. Arbutinum non finditur. Biotinae necessariae ad crescentiam. Materia amylo similis non producitur. Maxima temperatura crescentiar : 23-33°. Habitat in solani lycopersici malum corruptum, Fujisawa, Japonia. Growth in malt extract : After 3 to 7 days at 28°, cells are short to long oval, or elongate, sometimes irregularly shaped, (1.0-5.0) x (2.0-20.0) j, and single, in pairs, or in short chains. A ring is formed. Pellicle is formed by one-day incubation at 28°, then become yellowish white, dull, thick, and creep- ing after 3 to 4 days. Reproduction proceeds by multilateral budding. Growth on malt agar : After 2 days at 28°, the culture is ceracous, slightly raised, has a smooth surface and an undulate margin, and old culture may change to pale yellowish white, opaque to cretaceous, vesicular or rugose surface and erose margin.