INTERNATIONALJOURNAL OF SYSTEMATICBACTERIOLOGY, Apd 1980, p. 514-519 Vol. 30, No. 2 0020-7713/80/02-0514/06$02.00/0

Pichia cellobiosa, cariosilignicola, and Candida succiphila, New Species of Methanol-Assimilating

J. D. LEE AND K. KOMAGATA Institute of Applied Microbiology, university of Tokyo, Bunkyo-ku, Tokyo 113, Japan

In the course of a taxonomic study of methanol-assimilatingyeasts, a number of isolates were obtained from various natural habitats in Japan, and from these three new species, cellobiosa, Candida cariosilignicola, and Candida succiphila, have been described. P. cellobiosa differs from the related species Pichia castiZlae with respect to fermentation and assimilation of sugars, biotin requirement for growth, and lower maximum temperature for growth. C. carios- ilignicola differs from the related species Torulopsis apicola and Torulopsis bombicola with respect to assimilation of sugars and proton magnetic resonance spectra of cell wall mannans. C. succiphila is similar to C. cariosilignicola but differs from it with respect to fermentation and assimilation of sugars, lower maximum temperature for growth, higher deoxyribonucleic acid base composi- tion, and proton magnetic resonance spectra of cell wall mannans. The type strains of these new species are KL 1 (=IAM 12481), KL 24 (=IAM 12484),and KL 30 (=IAM 12489), respectively. Cultures of the type strains have been deposited in the Institute of Applied Microbiology Culture Collection Center, University of Tokyo, Tokyo, Japan, and the InsetUte for Fermentation, Osaka, Japan.

Since Ogata et al. (16) reported a methanol- inoculated into 10 ml of medium; incubation was at assimilating in 1969, a number of such 30" C with shaking. The enrichment culture technique yeasts have been described (1-3,8,9,11,14, 15, was used for isolation, and pure cultures were obtained 17, 18, 21, 23, 28). Hazeu et al. (7) examined by the conventional streaking technique. The strains isolated and their sources are listed in Table 1. methanol-assimilation in type strains preserved Characterization of yeasts. The methods used to at the Centraalbureau voor Schimmelcultures characterize the isolates were primarily those reported and reported assimilation by the type strains of by Wickerham (24) and Lodder (12). Vitamin require- 15 species in the genera Hansenula, Pichia, ments were determined by the method of Komagata Torulopsis, and Candida. However, the sys- and Nakase (10). Deoxyribonucleic acid was isolated tematics of methanol-assimilating yeasts have and purified by the procedure of Cryer et al. (4), and not been studied in detail. Therefore, we at- deoxyribonucleic acid base compositions were calcu- tempted to isolate methanol-assimilatingyeasts lated from thermal denaturation temperatures by the from a wide variety of natural sources in Japan formula of Marmur and Doty (13). The ubiquinone systems were determined by the method of Yamada in order to compare them taxonomically with and Kondo (25). Isolations and purifications of man- the presently described methanol-assimilating nans from yeast cells were carried out as previously yeast species. During the investigation, 10 of the reported by Gorin and Spencer (5). Proton magnetic new isolates were identified as belonging to three resonance (PMR) spectra of the mannans were ob- new species, which are named and described tained by using a JNM-4H 100 spectrometer equipped here. A taxonomic study on methanol-assirnilat- with a temperature control unit; these spectra were ing yeasts, including the new species named and determined from 0.4 ml of a D20 solution contain- described here, will be reported in the future. ing 80 mg of mannan at 70°C, using 3-(tri- methylsily1)propane sulfonic acid (sodium salt) as an internal standard. MATERIALS AND METHODS Isolation of yeasts. Yeasts were isolated from RESULTS AND DISCUSSION various sources by using a methanol medium contain- Three strains (KL 1, KL 2, and KL 5) form ing the following: (NH4 )2S04,3g; KH2PO+4 g; MgS04 - hat-shaped ascospores and assimilate galactose, 7H20, 0.5 g; NaC1, 0.2 g; ferric citrate, 3 mg; CaCIz- 2H20, 3 mg; MnSO4*4H20,0.5 mg; ZnSOs-7H20,0.5 L-sorbose, cellobiose,and a considerable number mg; CuS04- 5H2 0,0.05 rng; yeast extract, 0.2 g; meth- of other sugars. They resemble Pichia castillae anol, 10 nll; chloramphenicol, 50 mg; and distilled (19) in these characteristics but differ from it in water, 1,OOO ml. The pH was 4.5 or was adjusted to 6.6 that they ferment and cellobiose, do not with NaaHP04. About 1 g of source material was assimilate maltose, melibiose, or raffinose, re- 514 VOL. 30,1980 NEW SPECIES OF METHANOL-ASSIMILATING YEASTS 515

TABLE1. Sources of the strains studied wall mannan. Mating was not found in combi- Strain Source nations of these six strains, which are here placed in a new species, Candida succiphila. KL 1 Soil, Chiba Prefecture KL 2 Soil, Chiba Prefecture Descriptions of these three newly recognized KL 5 Soil, Metropolitan Tokyo species follow. KL 24 Decayed timbers, Yamanashi Pre- Pichiu cellobiosu sp. nov. (cel.lo.bi.o'sa. fecture M.L. adj. cellobiosus pertaining to cellobiose, a KL 14 Soil, Yamanashi Prefecture sugar derived from cellulose.) This description KL 16 Sap of peach tree, Yamanashi Pre- is based on strains KL 1, KL 2, and KL 5, which fecture possess the Same characteristics. KL 21 Sap of peach tree, Yamanashi Pre- Latin diagnosis. In medio liquid0 cellulae fecture subovoideae aut ovoideae, (1.8-4.5) x (2.7-5.4) KL 22 Peach, Yamanashi Prefecture pm, singulae aut binae. Sedimentum formatur. KL 30 Sap of peach tree, Yamanashi Pre- fecture Cultura in agaro flavoglauca, glabra, butyrosa, KL 31 Peach, Yamanashi Prefecture margine undulato. Pseudomycelium non forma- tur. Asci inconjugati fiunt, habentes 3 aut 4 sporos pileiformos in quoque asco, asci rumpun- quire biotin for growth, and have a lower maxi- ter. Species homothallica. mum temperature for growth. Since most Pichia D-G~UCOS~~et cellobiosum (lente) fermentan- species with hat- or Saturn-shaped ascospores tur at non D-galactosum, saccharosum, malto- possess Q8 (26) and most of the species in the sum, lactosum, raffhosum, melibiosum, nec tre- Hansenula possess Q7 (26), strains KL 1, halosum. KL 2, and KL 5 seem to be unique in that they D-GIUCOSW,D-gdaCtOSUm, L-sorbosum, cel- possess a ubiquinone system composed of both lobiosum, trehalosum, D-xylosum, L-arabi- Q8 (major component) and Q7 (minor compo- nosum, D-arabinosum, D-ribosum, methanolum, nent) and thus appear to be intermediate be- ethanolum, glycerolum, erythritolum, ribitolum, tween Pichia and Hansenula. These three galactitolum, mannitolum, glucitolum, sali- strains are here regarded as belonging to a new cinum, acidum succinicum (variabile),et acidum species, for which the name Pichia cellobiosa is citricum (variabile)assimilantur at non sacchar- proposed. osum, maltosum, lactosum, melibiosum, raffi- Strain KL 24 is asporogenous, has globose or nosum, melezitosum, inulinum, amylum solu- oval cells, does not form pseudomycelia or true bile, L-rhamnosum, a-methyl-D-glucosidum,DL- mycelia, and does not produce manifest carote- acidum lacticum, nec inositolum. noid pigments; furthermore, it does not assimi- Nitras kalicus non assimilatur. late inositol and produces no starch. These char- Biotinae necessariae ad crescentiam. acteristics fit those of the genus Torulopsis Maxima temperatura crescentiar: 35-36" C. Berlese (22). Yarrow and Meyer (27) proposed Proportio molaris G + C in acido deoxyribo- an amendment of the description of the genus nucleico: 36.9 mol%. Candidu Berkhout to allow for nonhyphal spe- Typus isolatus ex solum Chibaensis, depositus cies. In accordance with the amended descrip- in Institute of Applied Microbiology Culture tion, strain KL 24 was classified in the genus Collection Center, University of Tokyo (IAM Candidu. With respect to the fermentation of 12481), et Institute for Fermentation, Osaka, sugars, strain KL 24 resembles Torulopsis upi- Japan (IF0 1909). cola (6) and Torulopsis bombicola (20), but it Description. Growth in glucose-yeast ex- differs from them by its assimilation of trehalose, tract-peptone water: After 3 days at 25"C, the D-xylose, D-arabinose, and L-arabinose and its cells are oval or ovoid, 1.8 to 4.5 by 2.7 to 5.4 pm PMR spectrum of cell wall mannan (20). Strain (Fig. 1A). A pellicle is not formed. KL 24 is here regarded as a member of a new Growth on YM agar: After 1 month at 20°C, species, Candida cariosilignicola. a streak culture is brownish white, smooth, and Six strains (KL 14, KL 16, KL 21, KL 22, KL butyrous and has an undulate margin. 30, and KL 31) resemble strain KL 24 in mor- Growth on yeast morphology agar: After 1 phological and cultural characteristics, but they month at 20"C, a streak culture is yellowish differ from it and other related strains in the white, smooth with slight radial striations, and fermentation and assimilation of sugars. These butyrous and has an undulate margin. six strains cannot grow at 37OC, their deoxyri- Dalmau plate culture on potato dextrose agar: bonucleic acid base compositions are about 5% Pseudomycelia are not formed either aerobically higher than that of strain KL 24, and the PMR or anaerobically. spectra of their cell wall mannans are different Formation of ascospores: Ascospores are from-ae PMR spectrum of strain KL 24 cell formed readily on V-8 vegetable agar and YM 516 LEE AND KOMAGATA INT. J. SYST.BACTERIOL.

FIG. 1. Cells of P. cellobiosa, C. carwsilignicola, and C. succiphila. (A) Vegetative cells of P. cellobiosa grown in glucose-yeast extract-peptone water for 3 days at 25" C. (B) Ascospores of P. cellobiosa grown on YM agar for 7 days at 25" C. Arrow indicates hat-shaped ascospores. (C) Vegetative cells of C. cariosilignicola grown in glucose-yeast extract-peptone water for 3 days at 25" C. (0)Vegetative cells of C. succiphila grown in glucose-yeast extract-peptone water for 3 days at 25" C. The bars represent 70 pn in all illustrations.

agar after 5 to 7 days at 25°C. Strongly sporu- starch, L-rhamnose, a-methyl-D-glucoside, DL- lating cultures appear brownish red, presumably lactate, and inositol are not assimilated. because of the pigmentation of the ascospores. Assimilation of potassium nitrate: Negative. Ascus formation is usually preceded by the fu- Growth in vitamin-free medium: Negative. sion of independent cells, which frequently form Biotin is required for growth. conjugation tubes, or less frequently by soma- Growth on 50% (wt/wt) glucose yeast extract togamous autogamy. The asci contain three to agar: Negative. four rather small hat-shaped ascospores with Sodium chloride tolerance: Tolerates 9 to 10% short brims (Fig. 1B). The asci rupture at ma- NaCl. turity, releasing the spores soon after formation. Maximum temperature for growth: 35 to The strains examined are homothallic. 36" C. Fermentation: D-Glucose and cellobiose (la- Production of starchlike substance: Negative. tent) are fermented. D-Galactose, saccharose, Gelatin liquefaction: Negative. maltose, , raffinose, and melibiose are not Urease and extracellular deoxyribonuclease fermented. activity: Negative. Assimilation of carbon compounds: D-Glucose, Guanine plus cytosine content of nuclear de- D-galactose, L-sorbose, cellobiose, trehalose, D- oxyribonucleic acid: 36.9 mol%. xylose, L-arabinose, D-arabinose, D-ribose, meth- Ubiquinone system: Q8 (minor component, anol, ethanol, glycerol, erythritol, ribitol, galac- Q7). titol, D-mannitot, D-glUCitOl, and salicin are as- PMR spectrum of cell wall mannan: Group 9- similated. Assimilations of succinate and citrate d of Spencer and Gorin (Fig. 2). are variable. Saccharose, maltose, lactose, meli- Type strain: KL 1, which was isolated in 1977 biose, raffinose, melezitose, inulin, soluble from soil from Chiba Prefecture, Japan. Cultures VOL. 30,1980 NEW SPECIES OF METHANOL-ASSIMILATING YEASTS 517

m 0 . In m L?

6 5 4s 6 5 46 6 5 46 FIG. 2. PMR spectra of cell wall mannans. (A) P. cellobiosa. (l3) C. cariosilignicola. (C) C. succiphila.

of this strain have been deposited in the Institute iensis, depositus in Institute of Applied Micro- of Applied Microbiology Culture Collection Cen- biology Culture Collection Center, University of ter, University of Tokyo, with the accession Tokyo (IAM 12484), et Institute for Fermenta- number 12481 and in the Institute for Fermen- tion, Osaka (IF0 1910). tation, Osaka, under the number 1909. Description. Growth in glucose-yeast ex- Candida ca.riosilig&ola sp. nov. tract-peptone water: After 3 days at 25'C, the (ca.ri.o.si.lig.ni' co.la. L. adj. cariosus decayed, cells are globose or spherical, 1.8 to 2.7 by 1.8 to rotted; L. noun Zignum wood; L. -cola dweller; 3.0 pm (Fig. 1C). A pellicle is not formed. M.L. fem. n. carwsilignicola decayed-wood Growth on YM agar and yeast morphology dweller or inhabitant.) The description of this agar: After 1 month at 20°C, a streak culture is species is based on a single strain, KL 24. yellowish white, smooth, butyrous to slightly Latin diagnosis. In medio liquid0 cellulae mucoid, and glistening and has an undulate mar- rotundae aut ovoideae, (1.8-2.7) X (1.8-3.0) pm, gin. singulae, binae, aut in catenis brevis. Sedimen- Dalmau plate culture on potato dextrose agar: tum formatur. Cultura, in agar0 flavoglauca, gla- Pseudomycelia tire not formed either aerobically bra, butyrosa, vel seIflpimucosa, nitida, margine or anaerobically. undulato. Pseudomycelium non formatur. Formation of ascospores: Ascospores are not D-G~ucos~~,saccharosum, et raffinosum (1/ formed on YM agar, Gorodkowa agar, V-8 veg- 3) fermentantur at non D-galactosum,maltosum, etable agar, or Kleyn acetate agar. lactosum, cellobiosum, melibiosum, nec trehal- Fermentation: D-Glucose, saccharose, and raf- osum. fmose (113) are fermented. D-Galactose, maltose, D-G1ucosum, L-sorbosum, saccharosum, tre- cellobiose, lactose, and melibiose are not fer- halosum, raffinosum, D-XylOSUm, L-arabinosum, mented. D-arabinosum, D-ribosum, L-rhamnosum, meth- Assimilation of carbon compounds: D-Glucose, anolum, ethanolum, glycerolum, erythritolum, L-sorbose, saccharose, trehalose, raffinose, D-XY- ribitolum, galactitolum (tarde et exigue), D-man- lose, L-arabinose, D-arabinose, D-ribose, L-rham- nitolum, D-glucitolum, et acidum citricum assi- nose, methanol, ethanol, glycerol, erythritol, ri- milantur at non D-galactosum, maltosum, cello- bitol, galactitol (slow and weak), D-mannitol, D- biosum, lactosum, melibiosum, melezitosum, in- glucitol, and citrate are assimilated. D-Galactose, ulinum, amylum solubile, a-methyl+-glucosi- maltose, cellobiose, lactose, melibiose, melezi- dum, salicinum, DL-acidum lacticum, acidum tose, inulin, soluble starch, a-methyl-D-gluco- succinicum, nec inositolum. side, salicin, DL-lactate, succinate, and inositol Nitras kalicus assimilatur. are not assimilated. Biotinae necessariae ad crescentiam. Assimilation of potassium nitrate: Positive. Maxima temperatura crescentiar: 41-42' C. Growth in vitamin-free medium: Negative. Proportio molaris G + C in acido deoxyribo- Biotin is required for growth. nucleico: 35.1 mol%. Growth on 50% (wt/wt) glucose yeast extract Typus isolatus ex calinoso lignum Yamanash- agar: Negative. 518 LEE AND KOMAGATA INT. J. SYST.BACTERIOL.

Sodium chloride tolerance: Tolerates 6 to 7% Description. Growth in glucose-yeast ex- NaCL. tract-peptone water: After 3 days at 25"C, cells Maximum temperature for growth: 41 to are globose or spherical, 1.8 to 2.7 by 1.8 to 3.0 42OC. pm (Fig. 1D). A pellicle is not formed. Production of starchlike substance: Negative. Growth on YM agar and yeast morphology Gelatin liquefaction: Negative. agar: After 1 month at 20"C, a streak culture is Urease and extracellular deoxyribonuclease yellowish white, smooth, butyrous, and slightly activity: Negative. glistening with an undulate margin. Guanine plus cytosine content of nuclear de- Dalmau plate culture on potato dextrose agar: oxyribonucleic acid: 35.1 mol%. Pseudomycelia are not formed either aerobically Ubiquinone system: Q7. or anaerobically. PMR spectrum of cell wall mannan: Group 9- Formation of ascospores: Ascospores are not b of Spencer and Gorin (Fig. 2). formed on YM agar, Gorodkowa agar, V-8 veg- Type strain: KL 24, which was isolated in 1977 etable agar, or Kleyn acetate agar. from decayed timber in Yamanashi Prefecture, Fermentation: D-Glucose, D-galactose, and Japan. Cultures of this strain have been depos- cellobiose (latent) are fermented. Saccharose, ited in the Institute of Applied Microbiology maltose, lactose, raffinose, and melibiose are not Culture Collection Center, University of Tokyo, fermented. with the accession number 12484 and in the Assimilation of carbon compounds: D-Glucose, Institute for Fermentation, Osaka, under the D-gdaCtOSe, L-sorbose, cellobiose, trehalose, D- number 1910. xylose, L-arabinose, D-arabinose, D-ribose, L- Candida succiphila sp. nov. (suc.ci.phi'la. rhamnose, methanol, ethanol, glycerol, erythri- L. noun succus sap; Gr. adj, philus loving; M.L. tol, ribitol, gdactitol (latent or weak), D-manni- adj. succiphilus sap-loving.) The description of tol, D-glUCit01, and salicin are assimilated. Sac- this species is based on strains KL 14, KL 16, charose, maltose, lactose, melibiose, raffinose; KL 21, KL 22, KL 30, and KL 31, which possess melezitose, huh, soluble starch, a-methyl-D- the same characteristics. glucoside, DL-lactate, succinate, citrate, and ino- Latin diagnosis. In medio liquid0 cellulae sitol are not assimilated. rotundae aut ovoideae, (1.8-2.7) X (1.8-3.0) pn, Assimilation of potassium nitrate: Negative. singulae, binae aut in catenis brevis. Sedimen- Growth in vitamin-free medium: Negative. tum formatur. Cultura in agaro flavoglauca, gla- Biotin is required for growth. bra, butyrosa, nitida vel seminitida, margine un- Growth on 50% (wt/wt) glucose yeast extract dulato. Pseudomycelium non formatur. agar: Negative. D-G~ucos~~,D-galactosum, et cellobiosum Sodium chloride tolerance: Tolerates 7 to 8% (lente) fermentantur at non saccharosum, mal- NaCl. tosum, lactosum, raffinosum, melibiosum, nec Maximum temperatup for growth 35 to trehalosum. 36" C. D-GIucos~~,D-gdaCtOSum, L-sorbosum, cel- Production of starchlike substance: Negative. lobiosum, trehalosum, D-xylosum, L-arabi- Gelatin liquefaction: Negative , nosum, D-arabinosum, D-ribosum, L-rham- Urease and extracellular deoxyribonuclease nosum, methanolum, ethanolum, glycerolum, activity: Negative. erythritolum, ribitolum, galactitolum (lente aut Guanine plus cytosine content of nuclear de- exigue), D-glucitolum, et salicinum assimilantur oxyribonucleic acid: 40.9 mol%. at non saccharosum, maltosum, lactosum, meli- Ubiquinone system: Q7. biosum, raffinosum, melezitosum, inulinum, PMR spectrum of cell wall mannan: Group amylum solubile, a-methyl-D-glucosidum, DL- 9-a of Spencer and Gorin (Fig. 2). acidum lacticum, acidum succinicum, acidum Type strain: KL 30, which was isolated in 1977 citricum, nec inositolum. from the sap of a peach tree in Yamanashi Nitras kalicus Ron assimilatur. Prefecture, Japan. Cultures of this strain have Biotinae necessariae ad crescentiam. been deposited in the Institute of Applied Mi- Maxima temperatura crescentiar: 35-36" C. crobiology Culture Collection Center, University Proportio molaris G + C in acido deoxyribo- of Tokyo, with the accession number 12489 and nucleico: 40.9 mol%. in the Institute for Fermentation, Osaka, under Typus isolatus ex cerasus stemma Yamanash- the number 1911. iensis, depositus in Institute of Applied Micro- biology Culture Collection Center, University of ACKNOWLEDGMENT Tokvo (IAM 12489). et Institute for Fermenta- We thank S. Goto. Yamanashi Universitv. for his encour- tion: Osaka (IFO i9ii). agement and invaluable discussions. VOL. 30,1980 NEW SPECIES OF METHANOL-ASSIMILATING YEASTS 519

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