New Yeasts Capable of Assimilating Methanol*

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 microorganisms 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 assimilation 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 utilization 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-assimilating 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 medium was shown by 20 strains of yeast, 14 of which were globose, ovoid, cylindrical, or elongate in shape (Fig. 1A), and formed well-developed pseudomycelia, 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, inulinum, dextrinum, inositolum, maltosum, rafnosum nec a-methyl-D-glucosidum. 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, melezitosum, D-arabinosum, dulcitolum, a-methyl-D-glucosidum, potassium gluconatum, 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. Growth on methanol medium (medium D) : Giant colonies are pale yellow, opaque, dull, umbilicate, and have a rough or rugose surface. Slide culture : Well-developed pseudomycelia are formed. Blastospores are short oval to oval, and occur singly, in chains, or in verticils. Sporulation : None. Fermentation : Using biotin- and thiamine-containing medium, glucose is fermented, but galactose, sucrose, melibiose, lactose, inulin, dextrin, inositol, maltose, raffinose, and a-methyl-glucoside are not fermented. Assimilation of carbon compounds : Glucose + D-Ribose + Galactose - L-Rhamnose + L-Sorbose - Ethanol + Sucrose- Glycerol + Maltose- Erythritol+ Cellobiose- D-Glucitol+ Trehalose + D-Mannitol + Lactose- Ribitol+ 1972 Methanol-assimilating Yeasts 301

Melibiose- Galactitol- Raffinose - a-Methyl-D-glucoside - Melezitose- Salicin+ Inulin + DL-Lactic acid + Soluble starch + Succinic acid + D-Xylose + Citric acid - L-Arabinose + Inositol - D-Arabinose - Potassium gluconate - Calcium 2-ketogluconate- Methanol as a sole source of carbon : Good growth Assimilation of potassium nitrate : Positive Splitting of arbutin : Negative Vitamin stimulating growth : Biotin Production of carotenoid pigments : Negative Production of starch-like compounds : Negative Gelatin liquefaction : Negative Reaction in litmus milk: Negative Sodium chloride tolerance : 10% Optimum temperature for growth : 23 to 33°, very weak growth at 37° Optimum pH for growth : 3.0 to 6.0 Source : Rotten tomato, Fujisawa, Japan, 1968 According to the system of LODDER (15), the physiological and morpho- logical properties of this yeast seem to be similar to Candida boidinii. How- ever, some differences were found between this and C, boidinii in the carbon assimilation patterns and maximum temperature of growth, as shown in Table 3. Because of these facts this yeast is not cerrelated with C. boidinii. It was regarded as a new species of Candida and we propose to name it Candida met hanolica.

2. Torulopsis methanolovescens OKI et KouNO sp. nov. Strains : No. 1 (FERM No. 1107), 2, 3, 4, 5, and 6. Cellulae in extracto malti, globosae vel braviovoideae, (1.2-2) x (1.5-3) i, singulae aut binae. Pellicula non formatur. Cultura in agare (Post sex dies, 28°), cremea, nitida, mucosa, elevata et laevis. Pseudomycelium non formatur. Fermentatis nulla. In medio minerali D-glucosum, inulinum, amylum solbile, D-xylosum, L-arabinosum, D-ribosum, ethanolum, erythritolum, adonitolum, D-mannitolum, D-sorbitolum, acidum DL-lacticum, acidum succinicum et methanolum assimilantur at non D-galactosum, L-sorbosum, saccharosum, maltosum, cellobiosum, trehalosum, lactosum, melibiosum, rafbnosum, melezitosum, D- arabinosum, L-rhamnosum, glycerolum, dulcitolum, a-methyl-D-glucosidum, salicinum, potassium gluconatum, calcium 2-ketogluconatum, acidum citricum nec inositolum. Nitras kalicus non assimilatur. Arbutinum finditur. Biotinae et thiaminae necessariae ad crescentiam. Assimilatio ad methanolum esse abundat. Materia amylo similis non producitur. Maxima temperatura cres- 302 OK!, KouNo, KITAI, and OZAKI VOL. 18

Table 3. Comparison of physiological characteristics of the methanol-assimilating yeasts belonging to Candida with C. boidinii.

centiar ; 23-33°. Habitat in Rhododendron indici fibs siccatus, Tsujido, Japonia. Growth in malt extract : After 3 to 7 days at 28°, cells are round, nearly round, or short oval, (1.2-2.0) x (1.5-3.0) p, and single or in pairs (Fig. 2A). A ring is weakly formed. Pellicle is not formed. Reproduction proceeds by multilateral budding. Growth on malt agar : After 1 week cultivation at 28°, giant colonies are vitreous, cream colored, raised to pulvinate, smooth surface and entire margin, then change in mucoid and capitate. Growth on methanol medium (medium D) : Giant colonies are pale yellow, pulvinate, and has a smooth surface and entire or occasionally undulate margin. Slide culture : No true mycelia or pseudomycelia are formed (Fig. 2B). Sporulation : None Fermentation : Not observed Assimilation of carbon compounds : 1972 Methanol-assimilating Yeasts 303

Fig. 2. Cells of Torulopsis methanolovescens No. 1. A : Cells grown on MY agar for 3 days at 25 B Slide culture on PDA agar for 4 days at 25°. 304 OKI, KouNo, KITAI, and OzAKI VOL. 18

Table 4. Comparison of physiological properties of the methanol-assimilating yeasts belonging to Torulopsis with T. inconspicua.

Methanol as a sole source of carbon : Good growth Assimilation of potassium nitrate : Negative Splitting of arbutin : Positive Biotin and thiamine are essentially required Production of carotenoid pigments : Negative Production of starch-like compounds : Negative Reaction in litmus milk: Negative Gelatin liquefaction : Negative Sodium chloride tolerance : 8% Optimum temperature for growth : 23 to 33°, weak growth at 37° Optimum pH for growth : 3.0 to 6.0 Source : Rotten flower of azalea, Tsujido, Japan, 1968. There were no species identical with this yeast in the system of LODDER (15). Although some of the biochemical characteristics of this yeast are similar to those of Torulopsis inconspicua, differences were found in the properties as follows : The isolate assimilates various carbon compounds very 1972 Methanol-assimilating Yeasts 305 well, as shown in Table 4, but T, inconspicua does not have the ability to assimilate inulin, soluble starch, D-xylose, D-ribose, erythritol, ribitol, D- mannitol, D-glucitol, or methanol. Furthermore, there are considerable differences in the maximum temperature and vitamin requirement for growth. Compared with the methanol-assimilating yeasts reported previously (10, 12, 13), this yeast was quite different from Kloeckera sp. No. 2201 (10, 12) in reproduction, shape of the cell, and biochemical properties such as fermentation, splitting of arbutin, and the behavior on the assimilation of carbon compounds and nitrate, and we cannot prove the identity of this yeast with T. glabrata (13) through lack of taxonomical studies. We propose to name it Torulopsis methanolovescens because it strongly utilizes methanol.

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